CN110945126A

CN110945126A - Method for multiplex detection of alleles associated with corneal dystrophy

Info

Publication number: CN110945126A
Application number: CN201880033645.1A
Authority: CN
Inventors: C.赵-沈
Original assignee: Avellino Lab USA Inc
Current assignee: Avellino Lab USA Inc
Priority date: 2017-04-10
Filing date: 2018-04-10
Publication date: 2020-03-31
Anticipated expiration: 2038-04-10
Also published as: US20200040397A1; AU2018250570A1; KR20200017386A; EP3610015A1; JP2020516280A; KR102701188B1; JP2023116533A; US11905560B2; JP7303113B2; CA3059591A1; EP3610015A4; WO2018191304A1; CN110945126B

Abstract

The present disclosure provides a method for detecting corneal dystrophy in a subject comprising a reaction mixture comprising one or more labeled probes comprising a mutated TGFBI nucleotide sequence; the reaction mixture further comprises at least one amplification primer pair for amplifying the TGFBI gene sequence of a biological sample from the subject; and detecting 1,2, 3,4, 5 or 6 mutations selected from the group consisting of G623D, M502V, R124S, a546D, H572R and H626R mutations in the TGFBI gene, wherein detecting comprises detecting the one or more mutations using a labeled detection probe. The invention also provides a reaction kit comprising the reaction mixture.

Description

Method for multiplex detection of alleles associated with corneal dystrophy

Technical Field

The present application relates generally to probes for detecting or diagnosing corneal dystrophy, and methods of detecting or diagnosing corneal dystrophy.

Background

Real-time PCR can be used to detect differences between nucleic acid sequences having substantially the same sequence. By using differentially labeled fluorescent nucleic acid probes, such as probes that bind to wild-type sequences and probes that bind to mutant sequences, single nucleotide changes in the human genome can be detected quickly and reliably. This resolution has been applied in medical diagnostics, where Single Nucleotide Polymorphisms (SNPs), i.e. single base changes found within coding and/or non-coding sequences of proteins, are associated with human diseases.

However, real-time PCR analysis is highly dependent on the collection and separation of high quality samples. Poor sample collection and/or separation requires the use of longer assay conditions and larger amounts of real-time PCR reagents, both of which result in increased cost and reduced productivity. Furthermore, failure of real-time PCR single nucleotide polymorphism detection assays may result in the need to collect additional samples, resulting in even greater loss of time and resources.

Thus, there is a great need for methods that result in improved sample collection and separation, which increase the overall success rate of the assay, reduce the reagents required for the assay, and reduce the need to subsequently collect additional samples. In addition, methods for real-time PCRSNP detection assays using smaller amounts of sample material will also reduce the challenges associated with the collection and isolation of high quality samples.

The cornea is the avascular clear tissue in the front of the eye that begins the process of focusing light onto the retina and accounts for about two thirds of the light intensity of the eye a number of genetic diseases affect the clarity of the cornea and are classified by the affected corneal layers as posterior, stromal or superficial autosomal dominant inheritance (AD), X-linked recessive inheritance (XR) and autosomal recessive inheritance (AR), and in many cases the loci of the disease have been located and pathogenic genes have been identified the most studied corneal dystrophies are those caused by autosomal dominant missense mutations in the transforming growth factor β -induced gene (TGFBI) located on chromosome 5q31.1, which encodes extracellular matrix proteins that are believed to play a key role in physiological and pathological responses by mediating cell adhesion, migration, proliferation and differentiation, to date, 62 hgtgi mutations have been reported in dyhuman gene mutations (fbke) resulting in a series of different corneal stroma proteins with amyloid and non-like proteins and a series of corneal dystrophy-corneal stroma-type mutations that have been documented in the corneal dystrophy-type hgtgfbi-type corneal dystrophy, which has been previously been reported by the two corneal dystrophy-type nutritive mutations that have been reported on corneal stroma and corneal stroma, and corneal dystrophy, which have been reported by beckheielli-type corneal dystrophy.

Laser in situ keratomileusis (LASIK) is a surgical procedure that provides vision correction for myopia (nearsightedness), hyperopia (farsightedness), and astigmatism. A thin flap in the corneal epithelium is cut and folded, and the exposed stromal layer is reshaped by a laser to change its corneal focusing power. Small incision lensectomy (SMILE) is a less invasive procedure to correct myopia. A small incision is made in the epithelial layer by laser, and a small piece of stroma (lens) is removed to reshape the stroma. Laser optical keratectomy (PRK) and light therapeutic keratectomy (PTK) surgery affect vision correction or treat a variety of ocular diseases by removing haze and surface irregularities from the corneal surface. These invasive corneal surgeries cause wounds in the stromal layers, which leads to upregulation of TGFBI expression, leading to corneal amyloid deposition in individuals carrying TGFBI mutations, leading to pathologies associated with corneal dystrophy. LASIK is contraindicated in individuals with Granular Corneal Dystrophy (GCD). Commercially available genetic tests can detect the five most common mutations associated with the five more common types of corneal dystrophy within the TGFBI gene: granular corneal dystrophy type 2R 124H, latticed corneal dystrophy type 1R 124C, Reis-Buckler corneal dystrophy R124L, granular corneal dystrophy type 1R 555W, Thiel-Behnke corneal dystrophy R555Q. These five mutant gene tests were originally designed for the korean and japanese population, and most of TGFBI corneal dystrophy cases were diagnosed as GCD2 caused by the R124H mutation. In korea and japan, this test is mainly used as a screening tool before refractive surgery. However, in the united states and europe, this test is used both to screen refractive surgical candidates and as a confirmatory test for clinical diagnosis of corneal dystrophic diseases.

In view of the above background, there is a need in the art to examine the prevalence of different TGFBI mutations in various populations and geographic locations to improve genetic testing for different populations worldwide.

Disclosure of Invention

In one aspect, the present disclosure provides a reaction mixture for detecting corneal dystrophy in a subject, the reaction mixture comprising a labeled probe comprising a sequence selected from the group consisting of SEQ ID NOs: 25-30, 36 and 54. The reaction mixture may further comprise a corresponding labeled probe comprising a sequence selected from the group consisting of seq id NOs: 19-24, 33 and 50. In some embodiments, the label probe consists of a sequence selected from SEQ id nos: 25-30, 36 and 54; and/or the corresponding labeled probe consists of a nucleotide sequence selected from the group consisting of SEQ ID NO: 19-24, 33 and 50. In other embodiments, the reaction mixture comprises a labeled TGFBIG623D probe comprising the nucleotide sequence set forth in SEQ ID NO: 33 or 36; and a labeled TGFBI M502V probe comprising SEQ ID NO: 24 or 30. In still other embodiments, the labeled TGFBI G623D probe comprises SEQ id no: 36; and the labeled TGFBI M502V probe comprises SEQ ID NO: 30.

In some embodiments, the labeled probe is fluorescently labeled. In other embodiments, each of the labeled probes comprises a different probe. In still other embodiments, each of the labeled probes comprises a different probe independently labeled with VIC, FAM, ABY, or JUN.

In some embodiments, the reaction mixture further comprises at least one amplification primer pair for amplifying TGFBI gene sequences from a biological sample from a subject. In other embodiments, the reaction mixture comprises (a) a corresponding forward primer comprising a sequence selected from the group consisting of SEQ ID NOs: 7-12 and 41; and (b) a corresponding reverse primer comprising a sequence selected from SEQ ID NOs: 13-18 and 47. When the reaction mixture comprises a nucleic acid comprising SEQ ID NO: 33 or 36 and a labeled TGFBI G623D probe comprising the nucleotide sequence of SEQ ID NO: 24 or 30, the reaction mixture may further comprise (a) a corresponding forward primer comprising the nucleotide sequence of SEQ ID NO: 10 and 12; (b) a corresponding reverse primer; and (b) a corresponding reverse primer comprising SEQ ID NO: 16 and 18.

In one aspect, the present disclosure provides a reaction kit comprising a reaction mixture described herein. In one aspect, the reaction kit comprises a reaction mixture comprising detection probes for G623D and M502V mutations in TGBI genes. In some embodiments, the reaction kit further comprises one or more detection probes for R124S, a546D, H572R, and H626R mutations in the TGBI gene. In one aspect, the present disclosure provides a reaction kit comprising a reaction mixture as described herein and one or more marker probes for one or more TGFBI mutations selected from R124S, a546D, H572R and H626R. In some embodiments, the one or more labeled probes are separate from the reaction mixture. In other embodiments, the one or more label probes are selected from the group consisting of SEQ id nos: 19. 25, 20, 26, 21, 27, 23, 29, 50 and 54. In still other embodiments, the reaction kit comprises a labeled TGFBI R124S probe comprising the sequence of SEQ id no: 19 or 25. In still other embodiments, the reaction kit comprises a labeled TGFBI a546D probe comprising the sequence of SEQ ID NO: 20 or 26. In still other embodiments, the reaction kit comprises a labeled TGFBI H572R probe comprising the sequence of SEQ id no: 21 or 27. In still other embodiments, the reaction kit comprises a labeled TGFBI H626R probe comprising the sequence of SEQ ID NO: 23. 29, 50 or 54. In still other embodiments, the reaction kit further comprises an additional amplification primer set. In still other embodiments, the reaction kit further comprises a third amplification primer set for amplifying the TGFBI gene comprising the R124S mutation, a fourth amplification primer set for amplifying the TGFBI gene comprising the a546D mutation, a fifth amplification primer set for amplifying the TGFBI gene comprising the H572R mutation, and/or a sixth amplification primer set for amplifying the TGFBI gene comprising the H626R mutation.

In one aspect, the present disclosure provides a method for detecting corneal dystrophy, comprising detecting one, two, three, four, five or six mutations selected from: G623D, M502V, R124S, a546D, H572R and H626R mutations in the TGFBI gene. In some embodiments, detecting comprises sequencing the TGFBI gene. In other embodiments, detecting comprises detecting the mutation using a labeled detection probe.

In one aspect, the present disclosure provides a method for detecting corneal dystrophy, the method comprising: (a-1) amplifying a first TGFBI gene sequence from a biological sample from a subject using a reaction mixture comprising at least a first amplification primer pair and a set of at least two detection probes; (B-1) hybridizing the first detection probe and the second detection probe in the group having at least two detection probes with the first TGFBI gene sequence having the G623D mutation and the second TGFBI gene sequence having the M502V mutation, respectively; and (C-1) detecting one, two or more mutations in the TGFBI gene sequence based on hybridization of the first and second detection probes to the first and second TGFBI gene sequences, respectively. In some embodiments, the method further comprises (a-2) amplifying a third TGFBI gene sequence from the biological sample, wherein the reaction mixture further comprises a third marker probe for a third TGFBI mutation selected from the group consisting of R124S, a546D, H572R, and H626R; (B-2) hybridizing the third label probe with the third TGFBI gene sequence; (C-2) detecting a mutation in the third TGFBI gene sequence based on hybridization of the third detection probe to the third TGFBI gene sequence.

Drawings

Fig. 1A shows a world map of reported cases with multiple TGFBI mutations. The bubbles on each region or country/region contain reported case information such as race, mutation, and case number. The figure shows TGFBI mutation cases reported worldwide except in regions of limited study capacity or difficult language publishing. There are few cases reported in south america, and nothing was reported in africa or russia. Fig. 1B shows a red arrow pointing into the uk as an example of the information contained in the bubble. The legend on the left shows the reported mutations, ethnicity, and total number of cases for each reported mutation.

Fig. 2 provides a comparison by geographic area. In 1600 reported cases, the original gene test with five mutations, six additional mutations, and the proposed extended 11 mutation sets was modeled. The detection rates of available genetic tests with five mutations between europe and asia are very close.

Figure 3 provides a table ranking the five most common mutations in reported cases from highest to lowest. In addition, it lists six additional cases of mutation from high to low.

FIG. 4 provides a table showing the theoretical results of available gene testing for R124C, R555W, R124H, R555Q, and R124L. This test detected 90% of the 68 TGFBI CD cohorts identified by the Moorfield corneal dystrophy study. The table also shows the results of six additional mutations identified by literature studies. They increased the detection rate by 7% and led to an overall detection rate of 97% in the uk.

Fig. 5A-5C provide exemplary sequences of targets, primers, and probes used in the examples.

Fig. 6A and 6B show the discrimination map results of example 4 using M502V and G623D TGFBI probes.

Detailed Description

I. Introduction to the design reside in

The present disclosure is based, at least in part, on the discovery of reaction mixtures, reaction kits to improve the detection of corneal dystrophy.

In asia, the reported prevalence of TGFBI corneal dystrophy is 870 in korea and 416 in china. Asia, which is highly myopic, Holden et al, predicted that by 2050, myopia prevalence was the highest in the asian population, 66.4% of all populations, compared to 49.8% of the global prevalence. Due to the high prevalence of myopia in these asian populations, the use of LASIK vision correction surgery has increased and is expected to continue to increase. Mutation testing is important in asian populations due to the known prevalence of TGFBI mutations and high myopia rates; subsequently, five mutant gene tests were initially introduced in the asian-pacific population.

Since the first description of TGFBI mutations as a cause of granular corneal dystrophy in Folberg et al 1988, our understanding and appreciation of the disease has steadily increased. The most common R124 and R555 mutations are well documented, and additional mutations are being examined more closely to understand the next level of common variants. The present disclosure provides a review of literature reports on various TGFBI corneal dystrophies to understand the prevalence of the disease. The global prevalence of this disease is unknown; however, the consequences of this disease are debilitating. The ultimate treatment is corneal transplantation, and the recurrent nature of this disease often requires subsequent corneal transplants, which are both traumatic and costly to both the patient and the ophthalmologist. Therefore, prevention and pre-screening by molecular diagnostic tests to detect mutations is critical.

In some embodiments, it is an object to provide enhanced testing capabilities in a pre-screening test prior to refractive surgery. Another objective is to narrow the gap between the detection rates obtained from genetic testing and clinical diagnosis.

Selection definition

The term "invention" or "present invention" as used herein is not intended to limit any particular embodiment of the invention, but applies generally to any and all embodiments of the invention described in the claims and specification.

As used herein, singular terms include plural meanings unless the context clearly dictates otherwise. Thus, for example, reference to "a method" includes one or more methods and/or steps of the type described herein, as will be apparent to those skilled in the art upon reading this disclosure.

As used herein, the term "polymorphism" and variants thereof refer to the presence of two or more alternative genomic sequences or alleles between or among different genomes or individuals. The term "genetic mutation" or "genetic variation" and variants thereof include polymorphisms.

As used herein, the term "single nucleotide polymorphism" ("SNP") and variants thereof refer to a site of one nucleotide that varies between alleles. Single Nucleotide Polymorphisms (SNPs) are single base changes or point mutations, but also include so-called "insertion/deletion" mutations (insertions or deletions of nucleotides), resulting in genetic variation between individuals. SNPs account for about 90% of all human genetic variations, occurring once every 100 to 300 bases in a 30 hundred million base human genome. SNPs can occur in coding or non-coding regions of the genome. SNPs in the coding region may or may not alter the amino acid sequence of the protein product. SNPs in non-coding regions may alter promoter or processing sites and may affect gene transcription and/or processing. Knowledge of whether an individual has a particular SNP in a target genomic region may provide sufficient information to develop diagnostic, prophylactic and therapeutic applications for a variety of diseases. In some embodiments, the present disclosure relates to the detection of SNPs in coding regions that alter the amino acid sequence, resulting in mutations in the amino acid sequence of the product from the TGBI gene. For example, the present disclosure relates to the detection of SNPs causing mutations in G623D, M502V, R124S, a546D, H572R, H626R, G623D, R124S, H403Q, R124C, and/or R124H in the TGFBI gene.

The term "primer" and variants thereof refer to an oligonucleotide that serves as a point of initiation of DNA synthesis in a PCR reaction. The primer is typically about 15 to about 35 nucleotides in length and hybridizes to a region complementary to the target sequence.

The term "probe" and variants thereof (e.g., detection probes) refers to an oligonucleotide that hybridizes to a target nucleic acid in a PCR reaction. The target sequence refers to the region of nucleic acid to be analyzed and contains the polymorphic site of interest.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, various embodiments of the methods and materials are specifically described herein.

Reaction mixture

In one aspect, the present disclosure provides a reaction mixture for detecting corneal dystrophy in a subject, the reaction mixture comprising a detection probe for detecting a mutation in TGBI. In some embodiments, the detection probe detects a SNP that causes an amino acid mutation described herein. In one aspect, the present disclosure provides a reaction mixture for detecting corneal dystrophy in a subject, the reaction mixture comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 25-30, 36 and 54. The reaction mixture further comprises a corresponding labeled probe comprising a sequence selected from the group consisting of SEQ ID NOs: 19-24, 33 and 50. In some embodiments, the labeled probe consists of a sequence selected from SEQ ID NOs: 25-30, 36 and 54; and/or the corresponding labeled probe consists of a nucleotide sequence selected from the group consisting of SEQ ID NO: 19-24, 33 and 50. In other embodiments, the reaction mixture comprises a labeled TGFBI G623D probe comprising seq id NO: 33 or 36; and a labeled TGFBI M502V probe comprising SEQ ID NO: 24 or 30. In yet other embodiments, the labeled TGFBI G623D probe comprises SEQ ID NO: 36; and the labeled TGFBI M502V probe comprises SEQ ID NO: 30.

In some embodiments, the reaction mixture further comprises at least one amplification primer pair for amplifying the TGFBI gene sequence from a biological sample of the subject. In other embodiments, the reaction mixture comprises (a) a corresponding forward primer comprising a sequence selected from the group consisting of SEQ ID NOs: 7-12 and 41; and (b) a corresponding reverse primer comprising a sequence selected from SEQ ID NOs: 13-18 and 47. When the reaction mixture comprises a nucleic acid comprising SEQ ID NO: 33 or 36 and a labeled TGFBI G623D probe comprising the nucleotide sequence of SEQ ID NO: 24 or 30, the reaction mixture may further comprise (a) a TGFBI M502V probe comprising the nucleotide sequence of SEQ ID NO: 10 and 12, respectively; (b) comprises the amino acid sequence of SEQ ID NO: 16 and 18, respectively.

In some embodiments, the labeled probe is fluorescently labeled. In other embodiments, each of the labeled probes comprises a different probe. In still other embodiments, each of the labeled probes is independently labeled with VIC, FAM, ABY, or JUN.

IV. diagnostic kit

In one aspect, any or all of the reagents described herein are packaged into a diagnostic kit. Such kits include any combination of any and/or all of the primers, probes, buffers, and/or other reagents described herein.

In one aspect, the present disclosure provides a reaction kit comprising a primer set, detection probes and/or reagents for detecting R124S, a546D, H572R, H626R, G623D and M502V mutations in TGBI genes. In one aspect, the present disclosure provides a reaction kit comprising a primer set, a detection probe and/or a reagent for detecting G623D and M502V mutations in TGBI genes together with a separate reaction mixture comprising a combination of primer set, probe and/or reagent for detecting G623D and M502V. In some embodiments, the reaction kit further comprises one, two, three or four primer sets, detection probes and/or reagents for detecting one, two, three or four TGFBI mutations selected from R124S, a546D, H572R and H626R. In other embodiments, the reaction kit further comprises one, two, three, four or five primer sets, detection probes and/or reagents for detecting one, two, three, four or five TGFBI mutations selected from G623D, R124S, H403Q, R124C and R124H.

In one aspect, the present disclosure provides a reaction kit comprising the above-described reaction mixture and one or more additional reagents. In some embodiments, the reaction kit further comprises one, two, three or four primer sets, labeled probes and/or reagents for detecting one, two, three or four TGFBI mutations selected from R124S, a546D, H572R and H626R. In some embodiments, one, two, three or four primer sets, labeled probes and/or reagents for detecting one, two, three or four TGFBI mutations selected from R124S, a546D, H572R and H626R are separate from the reaction mixture in the kit. In other embodiments, the reaction kit comprises 1,2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 labeled probes selected from the group consisting of: comprises the amino acid sequence of SEQ ID NO: 19-24, 33, 50, 25-30, 36 and 54, respectively. In still other embodiments, the reaction kit comprises a labeled TGFBI R124S normal probe comprising SEQ ID NO: 19; and/or a labeled TGFBI R124S mutation probe comprising the amino acid sequence of SEQ id no: 25. In other embodiments, the reaction kit comprises a labeled TGFBI a546D normal probe comprising SEQ ID NO: 20; and/or a labeled TGFBI a546D mutation probe comprising SEQ ID NO: 26. In still other embodiments, the reaction kit comprises a labeled TGFBI H572R normal probe comprising the sequence of SEQ ID NO: 21; and/or the labeled TGFBI H572R mutation probe comprises SEQ ID NO: 27. In other embodiments, the reaction kit comprises a nucleic acid comprising SEQ ID NO: 23 or 50, and/or a TGFBI H626R normal probe comprising the nucleotide sequence of SEQ ID NO: 29 or 54, and a TGFBI H626R mutation probe. In other embodiments, the reaction kit does not include a kit in which the TGFBI G623D probe is stored alone or not mixed with the TGBI M502V probe. In other embodiments, the reaction kit further comprises an additional amplification primer set. In still other embodiments, the reaction kit further comprises a third amplification primer set for amplifying the TGFBI gene comprising the R124S mutation, a fourth amplification primer set for amplifying the TGFBI gene comprising the a546D mutation, a fifth amplification primer set for amplifying the TGFBI gene comprising the H572R mutation, and/or a sixth set of amplification primers for amplifying the TGFBI gene comprising the H626R mutation. Herein, the TGFBI gene comprising the R124S mutation may refer to a TGFBI gene comprising a SNP resulting in a R124S mutation in a TGBI protein product.

In other embodiments, the reaction kit further comprises one, two, three, four or five primer sets, detection probes and/or reagents to detect one, two, three, four or five TGFBI mutations selected from G623D, R124S, H403Q, R124C and R124H.

In some embodiments, the reagents in the kit are included as lyophilized powders. In some embodiments, the reagents in the kit are included as lyophilized powders with instructions for reconstitution. In some embodiments, the reagents in the kit are included as liquids. In some embodiments, the reagents are contained in plastic and/or glass vials or other suitable containers. In some embodiments, the primers and probes are all contained in separate containers within the kit. In some embodiments, the primers are packaged together in one container and the probes are packaged in another container. In some embodiments, the primer and probe are packaged together in a single container.

In some embodiments, the kit further comprises a control gDNA and/or DNA sample. In some embodiments, the control DNA sample included is a TGFBI sample having a G623 normal sequence and/or a TGFBI sample having an M502 normal sequence. In some embodiments, the control DNA sample included corresponds to the mutation detected, including R124S, a546D, H572R, and H626R. In some embodiments, a control DNA sample corresponding to TGFBI R124 normal and a mutant DNA sample corresponding to R124C, R124H, R124L, R555W, R555Q and/or H626P are included. In some embodiments, a control DNA sample corresponding to TGFBI R124 normal and a mutant DNA sample corresponding to R124C, R124H, R124L, R555W and/or R555Q are included. In some embodiments, a control DNA sample corresponding to TGFBI R124 normal and a mutant DNA sample corresponding to R124C, R124H, and/or R124L are included. In some embodiments, a control DNA sample corresponding to TGFBI R124 normal and a mutant DNA sample corresponding to R555W and/or R555Q are included. In some embodiments, a control DNA sample corresponding to TGFBI R124 normal and a mutant DNA sample corresponding to R124C are included. In some embodiments, a control DNA sample corresponding to TGFBI R124 normal DNA and a mutant DNA sample corresponding to R124H are included. In some embodiments, a control DNA sample corresponding to TGFBI R124 normal and a mutant DNA sample corresponding to R124L are included. In some embodiments, a control DNA sample corresponding to TGFBI R124 normal DNA and a mutant DNA sample corresponding to R555W are included. In some embodiments, a control DNA sample corresponding to TGFBI R124 normal and a mutant DNA sample corresponding to R555Q are included. In some embodiments, a control DNA sample corresponding to TGFBI R124 normal and a mutant DNA sample corresponding to H626P are included.

In some embodiments, the concentration of the control DNA sample is 5 ng/. mu.L, 10 ng/. mu.L, 20 ng/. mu.L, 30 ng/. mu.L, 40 ng/. mu.L, 50 ng/. mu.L, 60 ng/. mu.L, 70 ng/. mu.L, 80 ng/. mu.L, 90 ng/. mu.L, 100 ng/. mu.L, 110 ng/. mu.L, 120 ng/. mu.L, 130 ng/. mu.L, 140 ng/. mu.L, 150 ng/. mu.L, 160 ng/. mu.L, 170 ng/. mu.L, 180 ng/. mu.L, 190 ng/. mu.L, or 200 ng/. mu.L. In some embodiments, the concentration of the control DNA sample is 50 ng/. mu.L, 100 ng/. mu.L, 150 ng/. mu.L, or 200 ng/. mu.L. In some embodiments, the concentration of the control DNA sample is 100 ng/. mu.L. In some embodiments, the control DNA samples have the same concentration. In some embodiments, the control DNA samples have different concentrations.

In some embodiments, the kit may further comprise a buffer, such as GTXpress

A reagent mixture or any equivalent buffer. In some embodiments, the buffer comprises any buffer described herein.

In some embodiments, the kit may further comprise reagents for cloning, such as vectors (including, for example, the M13 vector).

In some embodiments, the kit further comprises reagents for purifying DNA.

In some embodiments, the kit further comprises instructions for using the kit to detect corneal dystrophy in a subject. In some embodiments, these specifications include various aspects of the protocols described herein.

Analysis of nucleic acids

In one aspect, the present disclosure provides a method for detecting corneal dystrophy, the method comprising detecting one, two, three, four, five or six TGFBI mutations selected from G623D, M502V, R124S, a546D, H572R and H626R in the TGFBI gene. In some embodiments, the method may further comprise detecting one, two, three, four or five TGFBI mutations selected from G623D, R124S, H403Q, R124C and R124H.

In some embodiments, detecting comprises sequencing the TGFBI gene. In other embodiments, detecting comprises detecting the mutation using a labeled detection probe.

In one aspect, the present disclosure provides a method for detecting corneal dystrophy, comprising: (a-1) amplifying a first TGFBI gene sequence from a biological sample of a subject using a reaction mixture comprising at least a first amplification primer pair and a set of at least two detection probes; (B-1) hybridizing the first detection probe and the second detection probe in the group having at least two detection probes with the first TGFBI gene sequence having the G623D mutation and the second TGFBI gene sequence having the M502V mutation, respectively; (C-1) detecting one, two or more mutations in the TGFBI gene sequence based on hybridization of the first detection probe and the second detection probe to the first TGFBI gene sequence and the second TGFBI gene sequence, respectively. In some embodiments, the method further comprises (a-2) amplifying a third TGFBI gene sequence from the biological sample, wherein the reaction mixture further comprises a third marker probe for a third TGFBI mutation selected from the group consisting of R124S, a546D, H572R, and H626R; (B-2) hybridizing the third marker probe with the third TGFBI gene sequence; and (C-2) detecting a mutation in the third TGFBI gene sequence based on hybridization of the third detection probe to the third TGFBI gene sequence.

In some embodiments, the methods herein further comprise isolating the genomic sample. In some embodiments, the method comprises providing a cell sample from the subject. In other embodiments, the subject may be a human. In some embodiments, the cells are collected by contacting the cell surface of the patient with a matrix capable of reversibly immobilizing the cells on the matrix.

The disclosed methods are applicable to a variety of cell types obtained from a variety of samples. In some embodiments, cell types used with the disclosed methods include, but are not limited to, epithelial cells, endothelial cells, connective tissue cells, skeletal muscle cells, endocrine cells, cardiac muscle cells, urinary cells, melanocytes, keratinocytes, blood cells, leukocytes. Buffy coat, hair cells (including, for example, hairy root cells), and/or salivary cells. In some embodiments, the cell is an epithelial cell. In some embodiments, the cells are subcapsular-perivascular (epithelial type 1), pallor (epithelial type 2), intermediate grade (epithelial type 3); dark (epithelial type 4), undifferentiated (epithelial type 5), and medullary (epithelial type 6). In some embodiments, the cell is a buccal epithelial cell (e.g., an epithelial cell collected using buccal exchange). In some embodiments, the cell sample used in the disclosed methods comprises any combination of the cell types identified above. In some embodiments, the provided cell is a buccal epithelial cell.

In some embodiments, it is advantageous to collect the sample in a non-invasive manner, and sample collection itself is done by anyone anywhere. For example, in some embodiments, the sample is collected at a physician's office, the subject's home, or at the site where LASIK surgery is or will be performed. In some embodiments, the patient's doctor, nurse or physician's assistant or other clinician collects the sample.

Various methods for analyzing SNPs of a sample (including, for example, but not limited to, genomic dna (gdna) samples) are known in the art, and may include PCR methods such as real-time PCR analysis, microarray analysis, hybridization analysis, and nucleic acid sequence analysis, as well as other various methods known to those skilled in the art of analyzing nucleic acid composition. See, e.g., molecular cloning (Triplex, Cold spring harbor laboratory Press, 2012) and the Manual of laboratory guides (genetics and genomics; molecular biology; 2003-.

a. Real-time PCR

With respect to the design of real-time PCR assays, several sections are adjusted, including DNA fragments (commonly referred to as amplicons) that flank the two primers and are subsequently amplified, the two primers used, and one or more detection probes.

Real-time PCR relies on the visual emission of fluorescent dyes conjugated to short polynucleotides (referred to as "detection probes"), which are related to genomic alleles in a sequence-specific manner. Real-time PCR probes that differ by only a single nucleotide can be distinguished in a real-time PCR assay by conjugating and detecting probes that fluoresce at different wavelengths. Real-time PCR finds use in detection applications (diagnostic applications), quantification applications, and genotyping applications.

Various related methods for performing real-time PCR are disclosed in the art, including relying on

The probe assay (U.S. Pat. Nos. 5,210,015 and 5,487,972, and Lee et al, Nucleic Acids Res.21: 3761-19, 6,1993), the molecular beacon probe (U.S. Pat. Nos. 5,925,517 and 6,103,476, and Tyagi and Kramer, nat. Biotechnol.14:303-8,1996), the self-probing amplicon (scorpion) (U.S. Pat. No. 6,326,145, and Whitcombe et al, nat. Biotechnol.17:804-7,1999), Amplissenor (Chen et al, Appl. environ. Microbiol.64:4210-6,1998), Amplifluor (U.S. Pat. No. 6,117,635, and Nazarko et al, Nucleic Acids Res. Res.25:2516,1997), the displacement probe (Li et al, Clcichl. 30: 30-1997, and the proximity PCR detection probe (Toquest. 22, 2000: 22, 2000-90, and Biotech. Pat. 22-2000-134).

In one aspect, the disclosure relates to the detection of SNPs causing mutations in G623D, M502V, R124S, a546D, H572R, H626R, G623D, R124S, H403Q, R124C, and/or R124H in the TGFBI gene. In some cases, real-time PCR may result in the detection of a variety of gene mutations, including, for example, but not limited to, SNPs. In some embodiments, real-time PCR is employed to detect SNPs in a particular gene candidate based on the use of intramolecular quenching of fluorescent molecules through the use of tethered quencher moieties. Thus, according to some exemplary embodiments, the real-time PCR method further comprises using molecular beacon technology. Molecular beacon technology utilizes hairpin-like molecules with internally quenched fluorophores whose fluorescence is restored by binding to the target DNA (see, e.g., R. et al Nat. Biotechnol.14:303-308, 1996). In some embodiments, increased binding of molecular beacon probes to accumulated PCR products is used to specifically detect SNPs present in genomic DNA.

One of the many suitable genotyping procedures is

And (4) carrying out allele identification determination. In some cases of this assay, an oligonucleotide probe labeled with a fluorescent reporter dye at the 5 'end of the probe and a quencher dye at the 3' end of the probe is utilizedAnd (3) a needle. The proximity of the quencher to the intact probe keeps the fluorescence of the reporter gene low. During the PCR reaction, the 5' nuclease activity of the DNA polymerase cleaves the probe and separates the dye and quencher. This results in an increase in the fluorescence of the reporter gene. The accumulation of PCR products was directly detected by monitoring the increase in fluorescence of the reporter dye. The 5' nuclease activity of the DNA polymerase cleaves the probe between the reporter and the quencher only when the probe hybridizes to the target and is amplified during PCR. Only when a specific SNP allele is present, the probe is designed to span the target SNP location and hybridize to the nucleic acid molecule.

By way of example, to amplify SNPs located on exon 4 of the TGFBI gene that are associated with Avellino corneal dystrophy, a forward PCR primer pair and a reverse PCR primer pair were constructed as described in U.S. patent publication No. 2012/0077200, the disclosures of which are incorporated herein by reference.

b. Real-time PCR cycling

Real-time PCR methods include multiple steps or cycles as part of the amplification process. These cycles include denaturing the double-stranded nucleic acid, annealing the forward primer, reverse primer and detection probe of the target genomic DNA sequence, and synthesizing (i.e., copying) the second-strand DNA from the annealed forward and reverse primers. The three step process is referred to herein as a loop.

In some embodiments, about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 cycles are employed. In some embodiments, about 10 to about 60 cycles, about 20 to about 50 cycles, or about 30 to about 40 cycles are employed. In some embodiments, 40 cycles are employed.

In some embodiments, the step of denaturing the double stranded nucleic acid is performed at a temperature of about 80 ℃ to 100 ℃, about 85 ℃ to about 99 ℃, about 90 ℃ to about 95 ℃ for about 1 second to about 5 seconds, about 2 seconds to about 5 seconds, or about 3 seconds to about 4 seconds. In some embodiments, the step of denaturing the double stranded nucleic acid is performed at a temperature of 95 ℃ for about 3 seconds.

In some embodiments, the step of annealing the forward primer, the reverse primer, and the detection probe of the target genomic DNA sequence is performed at about 40 ℃ to about 80 ℃, about 50 ℃ to about 70 ℃, about 55 ℃ to about 65 ℃ for about 15 seconds to about 45 seconds, about 20 seconds to about 40 seconds, about 25 seconds to about 35 seconds. In some embodiments, the step of annealing the forward primer, reverse primer and detection probe of the target genomic DNA sequence is performed at about 60 ℃ for about 30 seconds.

In some embodiments, synthesizing (i.e., replicating) second strand DNA from the annealed forward and reverse primers is performed at about 40 ℃ to about 80 ℃, about 50 ℃ to about 70 ℃, about 55 ℃ to about 65 ℃ for about 15 seconds to about 45 seconds, about 20 seconds to about 40 seconds, about 25 seconds to about 35 seconds. In some embodiments, the step of annealing the forward primer, reverse primer and detection probe of the target genomic DNA sequence is performed at about 60 ℃ for about 30 seconds.

In some embodiments, it was found that about 1 μ L, about 2 μ L, about 3 μ L, about 4 μ L, or about 5 μ L of a genomic DNA sample prepared according to the methods described herein was combined with only about 0.05 μ L, about 0.10 μ L, about 0.15 μ L, about 0.20 μ L, about 0.25 μ L, or about 0.25 μ L of 30X, 35X, 40X, 45X, 50X, or 100X real-time PCR assay mix and distilled water to form a PCR master mix. In some embodiments, the final volume of the PCR master mix is about 1.5. mu.L, about 2.5. mu.L, about 5. mu.L, about 6. mu.L, about 7. mu.L, about 8. mu.L, about 9. mu.L, about 0. mu.L, about 11. mu.L, about 12. mu.L, about 13. mu.L, about 14. mu.L, about 15. mu.L, about 16. mu.L, about 17. mu.L, about 18. mu.L, about 19. mu.L, or about 20. mu.L or more. In some embodiments, it was found that 2 μ L of the genomic DNA sample prepared as described above was combined with only about 0.15 μ L of the 40X real-time PCR assay mixture and 2.85 μ L of distilled water to form a PCR master mix.

Although exemplary reactions are described herein, one skilled in the art understands how to vary the temperature and time based on probe design. Further, any combination of the above times and temperatures are contemplated by the present methods.

PCR primers and primer design

In some embodiments, the primers are tested and designed in a laboratory setting. In some embodiments, the primers are designed by a computer-based electronic (in silico) method. The primer sequence is based on the amplicon sequence or target nucleic acid sequence to be amplified. Short amplicons generally replicate more efficiently and result in more efficient amplification than long amplicons.

In designing primers, the skilled artisan will appreciate the need to consider melting temperatures (T.sub.T) based on GC and AT content of the designed primer and secondary structure considerations (increased GC content may lead to increased secondary structure)_m(ii) a The temperature at which half of the primer-target duplex dissociates and becomes single-stranded, and is an indication of duplex stability; t is_mAn increase indicates an increase in stability). T can be calculated using a variety of methods known in the art_MAnd those skilled in the art will readily understand the various techniques for calculating T_MSuch a method of (a). Such methods include, for example, but are not limited to, on-line tools (such as T available on the world Wide Web promega. com/techserv/tools/biograph/calc 11.htm_MCalculator). Primer specificity is defined by its complete sequence in combination with the 3' end sequence (the part extended by Taq polymerase). In some embodiments, the 3' end should have at least 5 to 7 unique nucleotides that are not found anywhere else in the target sequence to help reduce false priming and false amplification product production. The forward and reverse primers typically bind to the target with similar efficiency. In some cases, alignment and primer design can be aided using tools (e.g., as NCBI BLAST, on the world wide web NCBI.

An additional aspect of primer design is primer complexity or linguistic sequence complexity (see, Kalendar R, et al (Genomics,98(2):137-&Chemistry 23: 263-274 (1999) and Y.L.Orlov, V.N.Potapov, compliance: an internet resource for analysis of DNA sequencecomplete, Nucleic Acids Res.32: W628-W633 (2004)), and is calculated as the sum of the observed ranges of 1 to L word lengths (xi) in the sequence divided by the sum of the expected (E) values for that sequence length. Some G-rich (and C-rich) nucleic acid sequences fold into stacked four-stranded DNA structures containing G tetraploids (see, world wide web quadruplex. In some cases, these tetraploids are formed by intermolecular association of two or four DNA molecules (dimerization of sequences comprising two G bases) or intermolecular folding of a single strand containing four guanine units (see, P.S.Ho, PNAS,91: 9549-; in some cases, due to its low complexity of language (for (TTAGGG)₄LC ═ 32%), these were eliminated by primer design.

These methods include various bioinformatic tools on CG content and melting temperature to model sequences with GC bias, (G-C)/(G + C), AT bias, (a-T)/(a + T), CG-AT bias, (S-W)/(S + W) or purine-pyrimidine (R-Y)/(R + Y) bias, and provide tools for determining language sequence complexity profiles. For example, the GC offset in a sliding window of n (where n is a positive integer), bases are calculated in steps of one base according to the formula (G-C)/(G + C), where G is the total number of guanines and C is the total number of cytosines for all sequences in the window (Y. Benita, et al, nucleic acids Res.31: e99 (2003)). A positive GC offset indicates G base excess, while a negative GC offset indicates C base excess. Similarly, other offsets in the sequence are calculated. In some embodiments, such methods and others are employed to determine primer complexity.

According to some non-limiting exemplary embodiments, exonuclease primers are used: (

Probe) for real-time PCR. In these embodiments, the primers utilize the 5' exonuclease activity of a thermostable polymerase (e.g., Taq) to cleave double-labeled probes present in the amplification reaction (see, e.g., Wittwer, C. et al, Biotechniques 22:130-138, 1997). Although complementary to the PCR product, the primer probe used in this assay is different from the PCR primer and is doubly labeled with a molecule capable of fluorescence and a molecule capable of quenching fluorescence. When the probe is intact, intramolecular quenching of the fluorescent signal within the DNA probe results in little to no signal. When the fluorescent molecule is released by exonuclease activity of Taq during amplification, the quenching is greatly reduced, resulting in an increase in the fluorescent signal. Non-limiting examples of fluorescent probes include 6-carboxy-fluorescein moieties and the like. Exemplary quenchers include Black Hole Quencher 1 moieties and the like.

Exemplary primers include, but are not limited to, those described herein. Primers for use in the disclosed methods are also found in U.S. patent publication No. 20120077200, which is incorporated herein by reference for all purposes. In some embodiments, PCR primers for use in the methods of the present disclosure include, but are not limited to, those listed below in the tables of fig. 5B and 5C, and are found for use in TGFBI gene detection. The biophysical parameters of each primer can be calculated using world wide web primer digital.com/tools/primer analyzer.

In some embodiments, the linguistic sequence complexity of the real-time PCR primers used in the disclosed methods is at least 70%, at least 72%, at least 75%, at least 77%, at least 80%, at least 82%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99%.

d. Design of detection probe and detection probe

Detection probes commonly used by those skilled in the art include, but are not limited to, hydrolysis probes (also referred to as hydrolysis probes)

Probes, 5' nuclease probes or double-labeled probes), hybridizationProbe and Scorpion primer (which binds primer and detection probe in one molecule). In some embodiments, the probe is designed to have a higher T than the primer_mTo facilitate efficient signal generation. Calculating T using any of a variety of methods known in the art_mAnd those skilled in the art will readily understand that such is used to calculate T_mThe various methods of (1); thus, such methods include, for example, those available in on-line tools, such as the calculator available on the world Wide Web promega.com/techserv/tools/bioomath/calc 11. htm.

In some embodiments, the detection probes contain various modifications. In some embodiments, the detection probes comprise modified nucleic acid residues such as, but not limited to, 2' -O-methyl ribonucleotide modifications, phosphorothioate backbone modifications, phosphorodithioate backbone modifications, phosphoramidate backbone modifications, methylphosphonate backbone modifications, 3' terminal phosphate modifications, and/or 3' alkyl substitutions.

In some embodiments, the affinity of the detection probe for the target sequence is increased as a result of the modification. Such detection probes include detection probes having increased length as well as detection probes containing chemical modifications. Such modifications include, but are not limited to, 2 '-fluoro (2' -deoxy-2 '-fluoro-nucleoside) modifications, LNA (locked nucleic acid), PNA (peptide nucleic acid), ZNA (zipper nucleic acid), morpholino, methylphosphonate, phosphoramidate, polycation conjugates, and 2' -pyrene modifications. In some embodiments, the detection probes contain one or more modifications, including 2' fluoro modifications (also known as 2' -deoxy-2 ' -fluoro-nucleosides), LNA (locked nucleic acids), PNA (peptide nucleic acids), ZNA (zipper nucleic acids), morpholinos, methylphosphonates, phosphoramidates, and/or polycation conjugates.

In some embodiments, the detection probe contains a detectable moiety, such as those described herein and any detectable moiety known to those of skill in the art. Such detectable moieties include, for example, but are not limited to, fluorescent labels and chemiluminescent labels. Examples of such detectable moieties may also include members of a FRET pair. In some embodiments, the detection probe contains a detectable entity.

Fluorescent markersExamples of (B) include, but are not limited to, ABY, JUN, AMCA, DEAC (7-diethylaminocoumarin-3-carboxylic acid), 7-hydroxy-4-methylcoumarin-3, 7-hydroxycoumarin-3, MCA (7-methoxycoumarin-4-acetic acid), 7-methoxycoumarin-3, AMF (4' - (aminomethyl) fluorescein), 5-DTAF (5- (4, 6-dichlorotriazinyl) aminofluorescein), 6-DTAF (6- (4, 6-dichlorotriazinyl) aminofluorescein), 6-FAM (6-carboxyfluorescein; also known as FAM, including

FAM^TM)、TAQMAN

5(6) -FAM cadaverine, 5(6) -FAM ethylenediamine, 5-FITC (FITC isomer I; fluorescein 5-isothiocyanate), 5-FITC cadaverine, fluorescein-5-maleimide, 5-IAF (5-iodoacetamido fluorescein), 6-JOE (6-carboxy-4 ',5' -dichloro-2 ',7' -dimethoxy fluorescein), 5-CR110 (5-carboxyrhodamine 110), 6-CR110 (6-carboxyrhodamine 110), 5-CR6G (5-carboxyrhodamine 6G), 6-CR6G (6-carboxyrhodamine 6G), 5(6) -rhodamine 6G cadaverine, 5(6) -carboxyrhodamine 6G ethylenediamine, 5-ROX (5-carboxy-X-rhodamine), 6-ROX (6-carboxy-X-rhodamine), 5-TAMRA (5-carboxytetramethylrhodamine), 6-TAMRA (6-carboxytetramethylrhodamine), 5-TAMRA cadaverine, 6-TAMRA cadaverine, 5-TAMRA ethylenediamine, 6-TAMRA ethylenediamine, 5-TMR C6 maleimide, 6-TMR C6 maleimide, TR C2 maleimide; TR cadaverine, 5-TRITC, the G isomer (tetramethylrhodamine 5-isothiocyanate), 6-TRITC, the R isomer (tetramethylrhodamine-6-isothiocyanate), dansylcadaverine (5-dimethylaminonaphthalene-1- (N- (5-aminopentyl)) sulfonamide), EDANS C2 maleimide, fluoroamine, NBD, and pyrromethene (pyrromethene) and derivatives thereof.

Examples of chemiluminescent labels include, but are not limited to, those used with the Southern Blot and Western Blot protocols (see, e.g., Sambrook and Russell, molecular cloning: A laboratory Manual (3 rd edition) (2001), incorporated herein by reference in its entirety). Examples include, but are not limited to- (2' -spiroadamantane) -4-methoxy-4- (3 "-phosphoryloxy) phenyl-1, 2-dioxane (AMPPD), acridinium esters, and adamantyl-stabilized 1, 2-dioxane and derivatives thereof.

In some embodiments, the labeled probe is used to hybridize within the amplified region during amplification. Probes may be modified to avoid them acting as primers for amplification. The detection probe may be labeled with two fluorescent dyes, one capable of quenching the fluorescence of the other. One dye is attached to the 5' end of the probe and the other dye is attached at an internal site, so quenching occurs when the probe is in a non-hybridized state.

Typically, real-time PCR probes consist of a dye pair (reporter and acceptor) that participates in Fluorescence Resonance Energy Transfer (FRET), whereby the acceptor dye quenches the emission of the reporter dye. In general, fluorescently labeled probes improve the specificity of amplicon quantification.

In view of the present disclosure, the real-time PCR used in some embodiments of the disclosed methods also includes the use of one or more hybridization probes (i.e., detection probes), as determined by one of skill in the art. By way of non-limiting example, such hybridization probes include, but are not limited to, one or more of those provided in the methods. Exemplary probes (e.g., HEX channel and/or FAM channel probes) are understood by those of skill in the art.

According to some exemplary embodiments, detection probes and primers are conveniently selected, for example, using electronic analysis of primer design software and cross-referencing available nucleotide databases of genes and genomes maintained at the National Center for Biotechnology Information (NCBI). In some embodiments, some additional guidelines may be used to select primers and/or probes. For example, in some embodiments, the primers and probes are selected so that they are close together but do not overlap. In some embodiments, the primers may have the same (or close T's)_M) (e.g., about 58 ℃ to about 60 ℃). In some embodiments, T of the probe_MRatio of T_MT selected by primer_MAbout 10 c higher. In some embodiments, the length of the probe and primer is selected to be about 17 to 39 bases equivalent. In some cases, the skilled artisan will select the appropriate primer and/or probeThese and other guidelines are used.

Probes useful in the methods of the invention include, but are not limited to, the following exemplary probes listed in fig. 5B and 5C.

Examples

Example 1: global document search

Interrogation of the HGMD database revealed 62 different TGFBI mutations. The HGMD database was used to identify papers in which these mutations were described in order to construct global profiles (fig. 1A and 1B). Each marker in the world map contains a summary of mutations reported in a particular region or country/region. Summary includes race, mutation and total number of cases reported for each mutation (fig. 1A). There was no significant difference in the distribution of mutations in a particular population or geographic region. There are few cases reported in south america, and nothing was reported in africa or russia. The map may be used to extract information for a particular country/region, such as london, indicated by the red arrow in fig. 1B.

Worldwide, 75% of TGFBI mutations reported in more than 1,600 cases consist of one of five mutations currently detected by available genetic tests. Although it is possible to issue new TGFBI mutations reported, in contrast, the most common TGFBI mutations found at codons R124 and R555 were not reported. Therefore, it is difficult to obtain an accurate assessment of the true global detection rate of TGFBI malnutrition in the literature.

Based on the ranking of the highest number of reported cases in our study, the effect on the detection rate of TGFBI mutations was evaluated by adding six mutations to the available gene test panel. The table of figure 3 shows the number of reported cases for each of the five most common mutations and six additional mutations proposed for the extension test. Notably, H626R is the fourth most prevalent mutation next to R124L. This finding supports the incorporation of this mutation into the extended group for the diagnosis of TGFBI corneal dystrophy. Although only 4 TGFBI corneal dystrophies associated with M502V were reported in the literature (supplementary material), a heterozygous mutation for M502V was detected in one sample. Therefore, it is included in the extended set.

From the literature reported cases, the addition of 6 new mutations in the existing group can increase global detection rates from 75% to 90% (fig. 2). The addition of additional mutations to the available gene tests theoretically resulted in a 32% increase in detection rate in south america and a 30% increase in north america. Both european and asian detection rates increased 13% and also benefited from the eleven mutation panel proposed (fig. 2).

Example 2: analysis of globally available genetic test data

Since 2008, over 600,000 samples have been tested worldwide by available genetic testing; most samples were from korea and japan, with the test being for pre-refractive surgical screening. Analysis of global test data showed that the detection rate in korea was about 15 out of 10,000, with a reported prevalence of 1 out of 870¹⁰In close proximity. The detection rate of TGFBI mutations in Japan (3 out of 10,000) is lower than that in Korea. In korea, this test is a routine screening for all candidates for refractive surgery, whereas in japan, patients are first subjected to a rigorous clinical examination, and only those patients for whom no corneal abnormality is detected submit samples for genetic testing.

Korean and japanese clinics/hospitals use genetic testing for screening purposes as it forms part of the refractive surgery practice guidelines. In the united states, some clinics/hospitals use this test for screening at the pre-operative exam of vision correction surgery, while others use it as confirmation of clinical diagnosis, or to rule out TGFBI mutations if the surgeon has any doubt about the defect noted in the patient's cornea. European clinics primarily utilize this test for this type of clinical validation.

Example 3: evaluation of expanded groups with six additional mutations

Few studies of populations with corneal dystrophy such as 2016UCLMoorfield performed Sanger sequencing of the entire TGFBI gene. This study provides us with a set of data based on which to evaluate the addition of six new mutation sites to increase the pointing rate of a given population. Briefly, the study consisted of 91 unrelated TGFBI corneal dystrophy cases, 68 of which were diagnosed as epithelial interstitial TGFBI-related dystrophies (RBCD, TBCD, LCD and r)GCD), 23 cases diagnosed as bilateral Epithelial Basement Membrane Dystrophy (EBMD)⁴. For the uk population, six TGFBI mutation groups were evaluated to determine whether these mutations were suitable for combination with the five mutant gene test. The data show that the detection rate in the uk population increased from 90% to 97% (table in figure 4). Other candidate mutations, such as V625D and a620D in the table of fig. 4, may be considered to increase the detection rate to almost 100%. This finding indicates that the inclusion of six additional mutations in the available gene tests, while increasing the index rate, still missed some of the important mutations found in the uk population.

16 of the 19 samples with clinical indications (negative with the original gene test) remained negative (84.2% of the total), while three tests in the expanded group were positive (15.7% of the total). WES results for maternal-child pairs with late clinical diagnosis of LCD were positive for the heterozygous TGFBI H626R mutation. Parallel real-time PCR testing showed the same heterozygous H626R mutation. The third sample was found to be heterozygous for M502V. Sanger sequencing confirmed the results. Subsequent patient history revealed very little corneal scarring on the patient's left cornea. There was no family history of corneal dystrophy or haze.

Based on evidence in the literature, the addition of six mutations to available genetic tests resulted in a 15% increase in detection rate. This is consistent with a 15.7% improvement in the detection of our sample cohort (3 out of 19 samples). No geographic or demographic differences were detected; therefore, six additional mutations newly proposed are suitable for use worldwide as an enhancement to current genetic testing. The new mutation greatly improves the mutation detection rate.

With respect to the presence of 6 additional mutations in the extension set, testing of 19 samples demonstrated that the extension gene test improved the detectability of TGFBI mutations.

Example 4: multiplex detection of mutations

First, for each mutation as shown in fig. 5B, a form 1(V1, version 1) primer, a VIC label probe having a normal sequence, and a FAM label probe having a mutant sequence were combined to detect the mutation. Detection of each of R124S, a546D, H572R, G623D, H626R, and M502V was successful. Next, for each of the a546D, H572R, and G623D mutations shown in fig. 5B, the V1 primer, the ABY label probe having a normal sequence, and the JUN label probe having a mutant sequence were combined to detect the mutation. Only the detection of the G623D mutation was successful. Third, for each of the R124S, H626R, and M502V mutations shown in fig. 5C, the form 2(V2) primer, a VIC marker probe having a normal sequence, and a FAM marker probe having a mutated sequence were combined to detect the mutations. Only the detection of H626R was successful. Fourth, for each of the a546D, H572R, and G623D mutations shown in fig. 5C, the V2 primer, the ABY label probe having a normal sequence, and the JUN label probe having a mutated sequence were combined to detect the mutation. Mutations were not correctly detected. Fifth, in a single reaction mixture, primers and probes are mixed to detect different combinations of mutations.

The following PCR master mix volume calculations and PCT conditions were used:

volume of TaqPath ProAmp Master mix; 2.5uL per test

Forward and reverse primers for M502V V1 primer, and volume of VIC and FAM probe mixture; 0.05uL per test

Forward and reverse primer volumes of G623D 20pM V1 primer: 0.05uL per test

G623D 50pM V1 ABY probe volume: 0.025uL per test

G623D 50pM V1 JUN probe volume: 0.025uL per test

Volume of water: 2.35uL per test

PCR fluorescence detection amplification cycle number and conditions:

cycle number: 40 cycles

Cycling conditions;

pre-PCR read (hold state): 60.0-01: 00 min

And (3) a heat preservation stage: 95.0-00: 20 min

And (3) circulating state: 40 cycles of 95.0 deg.C-00: 30 minutes

post-PCR read (hold phase): 60.0-01: 00 min

In addition to the primers and probes used for different combinations of mutations in a single reaction mixture, only the V1M 502V primer, the VIC and FAM probes with the V1G 623D primer, and the ABY and JUN probes successfully detected two mutations in a single reaction mixture, as shown in fig. 6A and 6B. The reagent combinations of R124S and a546D and H626R and H572R failed to detect mutations correctly.

The GRCh38.p7 homo sapiens transforming growth factor β induced (TGFBI), RefSeqGene on chromosome 5, NCBI reference sequence NG-012646.1 (SEQ ID NO: 61) are shown below.

The TGFBI gene protein product (β IG-H3 protein sequence; NCBI reference NG-012646.1) (SEQ ID NO: 62) is shown below.

All headings and section designations are used for clarity and reference purposes only and should not be construed as limiting in any way. For example, those skilled in the art will recognize the usefulness of combining various aspects from different headings as appropriate in view of the spirit and scope of the present invention as described herein.

All references cited herein are hereby incorporated by reference in their entirety and for all purposes to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

As will be apparent to those skilled in the art, many modifications and variations of this application can be made without departing from the spirit and scope of the invention. The specific embodiments and examples described herein are offered by way of example only, and the application is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled.

Sequence listing

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Chao-Shern, Connie

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<223> A546D Forward primer

<400>8

tctacacagt ctttgctccc aca 23

<210>9

<211>19

<212>DNA

<213> Artificial sequence

<220>

<223> H572R Forward primer

<400>9

ccaaggaact tgccaacat 19

<210>10

<211>18

<212>DNA

<213> Artificial sequence

<220>

<223> G623D Forward primer

<400>10

ttgccgagcc tgacatca 18

<210>11

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> H626R Forward primer

<400>11

ctgacatcat ggccacaaat gg 22

<210>12

<211>16

<212>DNA

<213> Artificial sequence

<220>

<223> M502V Forward primer

<400>12

ggaccgggtg ctgacc 16

<210>13

<211>19

<212>DNA

<213> Artificial sequence

<220>

<223> R124S reverse primer

<400>13

tccatctcag gcctcagct 19

<210>14

<211>19

<212>DNA

<213> Artificial sequence

<220>

<223> A546D reverse primer

<400>14

ctccgttctc ttggtggca 19

<210>15

<211>23

<212>DNA

<213> Artificial sequence

<220>

<223> H572R reverse primer

<400>15

cctccgctaa ccaggatttc atc 23

<210>16

<211>23

<212>DNA

<213> Artificial sequence

<220>

<223> G623D reverse primer

<400>16

tgcagaacat tggtgatgac atg 23

<210>17

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> H626R reverse primer

<400>17

ggaggctgca gaacattggt 20

<210>18

<211>18

<212>DNA

<213> Artificial sequence

<220>

<223> M502V reverse primer

<400>18

ctcccttcag gacatcca 18

<210>19

<211>15

<212>DNA

<213> Artificial sequence

<220>

<223> R124S VIC Probe (Normal)

<400>19

ctccgtgcgg tccgt 15

<210>20

<211>16

<212>DNA

<213> Artificial sequence

<220>

<223> A546D VIC Probe (Normal)

<400>20

ctcggaaggc ttcatt 16

<210>21

<211>18

<212>DNA

<213> Artificial sequence

<220>

<223> H572R VIC Probe (Normal)

<400>21

cctgaaatac cacattgg 18

<210>22

<211>16

<212>DNA

<213> Artificial sequence

<220>

<223> G623D VIC Probe (Normal)

<400>22

cacaaatggc gtggtc 16

<210>23

<211>16

<212>DNA

<213> Artificial sequence

<220>

<223> H626R VIC Probe (Normal)

<400>23

cgtggtccat gtcatc 16

<210>24

<211>17

<212>DNA

<213> Artificial sequence

<220>

<223> M502V VIC Probe (Normal)

<400>24

tgacagtccc cattggg 17

<210>25

<211>16

<212>DNA

<213> Artificial sequence

<220>

<223> R124S FAM Probe (mutation)

<400>25

tctccgtgct gtccgt 16

<210>26

<211>16

<212>DNA

<213> Artificial sequence

<220>

<223> A546D FAM Probe (mutation)

<400>26

ctcggaagtc ttcatt 16

<210>27

<211>18

<212>DNA

<213> Artificial sequence

<220>

<223> H572R FAM Probe (mutation)

<400>27

cctgaaatac cgcattgg 18

<210>28

<211>17

<212>DNA

<213> Artificial sequence

<220>

<223> G623D FAM Probe (mutant)

<400>28

ccacaaatga cgtggtc 17

<210>29

<211>14

<212>DNA

<213> Artificial sequence

<220>

<223> H626R FAM Probe (mutant)

<400>29

tggtccgtgt catc 14

<210>30

<211>17

<212>DNA

<213> Artificial sequence

<220>

<223> M502V FAM Probe (mutation)

<400>30

tgacagtccc cactggg 17

<210>31

<211>32

<212>DNA

<213> Artificial sequence

<220>

<223> A546D ABY Probe (Normal) -NN complementation

<400>31

cttggtggca gggctcggaa ggcttcattt gt 32

<210>32

<211>33

<212>DNA

<213> Artificial sequence

<220>

<223> H572R ABY Probe (Normal) -NN complementation

<400>32

cttgccaaca tcctgaaata ccacattggt gat 33

<210>33

<211>31

<212>DNA

<213> Artificial sequence

<220>

<223> G623D ABY Probe (Normal) -NN complementation

<400>33

catcatggcc acaaatggcg tggtccatgt c 31

<210>34

<211>32

<212>DNA

<213> Artificial sequence

<220>

<223> A546D JUN Probe (mutation) HH complementation (complement)

<400>34

cttggtggca gggctcggaa gtcttcattt gt 32

<210>35

<211>33

<212>DNA

<213> Artificial sequence

<220>

<223> H572R JUN Probe (mutation) HH complementation (complement)

<400>35

cttgccaaca tcctgaaata ccgcattggt gat 33

<210>36

<211>31

<212>DNA

<213> Artificial sequence

<220>

<223> G623D JUN Probe (mutation) HH complementation (complement)

<400>36

catcatggcc acaaatgacg tggtccatgt c 31

<210>37

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> R124S Forward primer

<400>37

gaccctggag tcgttggatc 20

<210>38

<211>24

<212>DNA

<213> Artificial sequence

<220>

<223> A546D Forward primer

<400>38

cgggaaggag tctacacagt cttt 24

<210>39

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> H572R Forward primer

<400>39

tgaaataccc caaggaactt g 21

<210>40

<211>18

<212>DNA

<213> Artificial sequence

<220>

<223> G623D Forward primer

<400>40

aggagcctgt tgccgagc 18

<210>41

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> H626R Forward primer

<400>41

ttgccgagcc tgacatcatg gc 22

<210>42

<211>16

<212>DNA

<213> Artificial sequence

<220>

<223> M502V Forward primer

<400>42

gttcacgatg gaccgg 16

<210>43

<211>18

<212>DNA

<213> Artificial sequence

<220>

<223> R124S reverse primer

<400>43

cccggcagct tcaccatc 18

<210>44

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> A546D reverse primer

<400>44

aagagtctgc tccgttctct t 21

<210>45

<211>24

<212>DNA

<213> Artificial sequence

<220>

<223> H572R reverse primer

<400>45

gccccgatgc ctccgctaac cagg 24

<210>46

<211>24

<212>DNA

<213> Artificial sequence

<220>

<223> G623D reverse primer

<400>46

cctggaggct gcagaacatt ggtg 24

<210>47

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> H626R reverse primer

<400>47

cacttacctg gaggcgcaga 20

<210>48

<211>18

<212>DNA

<213> Artificial sequence

<220>

<223> M502V reverse primer

<400>48

agcgattgtc tcccttca 18

<210>49

<211>27

<212>DNA

<213> Artificial sequence

<220>

<223> R124S VIC Probe (Normal)

<400>49

cgacttctcc gtgcggtccg tgtacag 27

<210>50

<211>16

<212>DNA

<213> Artificial sequence

<220>

<223> H626R VIC Probe (Normal)

<400>50

tggcgtggtc catgtc 16

<210>51

<211>17

<212>DNA

<213> Artificial sequence

<220>

<223> M502V VIC Probe (Normal)

<400>51

tccccattgg ggggggt 17

<210>52

<211>40

<212>DNA

<213> Artificial sequence

<220>

<223> R124S FAM Probe (mutation)

<400>52

cgacttcgac tttctccgtg ctgtccgtgt acaggtacag 40

<210>53

<211>16

<212>DNA

<213> Artificial sequence

<220>

<223> H626R FAM Probe (mutant)

<400>53

tggcgtggtc cgtgtc 16

<210>54

<211>17

<212>DNA

<213> Artificial sequence

<220>

<223> M502V FAM Probe (mutation)

<400>54

tccccactgg ggggggt 17

<210>55

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> A546D ABY Probe (Normal) -NN complementation

<400>55

agggctcgga aggcttcatt 20

<210>56

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> H572R ABY Probe (Normal) -NN complementation

<400>56

acatcctgaa ataccacatt gg 22

<210>57

<211>16

<212>DNA

<213> Artificial sequence

<220>

<223> G623D ABY Probe (Normal) -NN complementation

<400>57

cacaaatggc gtggtc 16

<210>58

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> A546D JUN Probe (mutation) HH complementation (complement)

<400>58

agggctcgga agtcttcatt 20

<210>59

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> H572R JUN Probe (mutation) HH complementation (complement)

<400>59

acatcctgaa ataccgcatt gg 22

<210>60

<211>16

<212>DNA

<213> Artificial sequence

<220>

<223> G623D JUN Probe (mutation) HH complementation (complement)

<400>60

cacaaatgac gtggtc 16

<210>61

<211>41924

<212>DNA

<213> Intelligent people

<400>61

agagggaaca gaagcatcta ggagagattt ggaaagaaca cctgcaggat cttggtgact 60

gattgcacgt gggggaccag agagcaggga caggcaaaac tgaatgcaag gtttccaacc 120

ttgagcggca ccacaggcaa gaatgaagaa atgaagaagg ggagctggac gaaagagcca 180

agggatttct gcattttgga atgaattgct gctgggtggt gtccatttcc ctgaaggcct 240

ttatcctacg tgcaagaaaa ctcgtgggaa gcagaggaaa ggcatgtgta agccaacaat 300

catctgtggg catccttcca ctaaagtatt tgaggtcagg caactaaagc aacctcaaaa 360

gtgcctctgg attcttctta gatattttag ctgagccaaa tcaatgaaac tctcatgaaa 420

aatcggtttc cctggaaaat gaaattgggt tctaaccaac aagtagcatt tggcaggccc 480

tgattaagaa agccagtgtt tggagaagtt gtgaaaacag ccaagtcatt taagaaacta 540

aacactgggg cctaatgcca ttctagggct gcgacggctg ttctgttccc atcaattgca 600

gagcccgaag cctcaagttt gttttaagtt cctgccatta caaacctgtc gattatccca 660

gcctcccttg cgggctttga aaagagagaa gaatggaagg tgactgtggc caatttcccc 720

tccctgtcca gtgtgtggaa gacactgaat atgcaactac tgaccttgtg cctgggcatc 780

ttgaaggtct tccacaaagt gagctgggcc tcagcggaag atgagagttc ctctgtggtc 840

acttcactgg tacacatttt caggtgtatt tcgtttcttc catgcctaca taaattgaat 900

cctctgttaa ccacctctga gctcatagct atttaacatg accctgtagt cctgtgcata 960

caaatcacct tgggatctgg tgaaaatgca gattcagtgg gtcttgggag gttgggaggt 1020

tataagattc cacgtttctt catgagagct agaaaaaata aataaataaa taaaaaattt 1080

ttaaattttc cacatttcta atgaactctg gggttgtgct gatgatgctg ttttgcagat 1140

cacattttga gtggcaagac tgtggaaaat ccttgagaaa tcaatccaaa atcccctaaa 1200

tggtactaca atcacacctt aatgttagta aactgagatg tttcttacct ttatttgtaa 1260

catggaaaaa acaattactg tatatgaagt accattctaa gttctgtgtg ttacacaagg 1320

gatggcaatt ttccccaaaa tttgattcac atcttttcat ttggatatct cttgccaaaa 1380

ctcacctttt tttctcccta gcaagtcttg gggagctgaa ttttaagagc tctttattta 1440

gctatatggt ggcctctgaa aatgattttg actgtatctt ctgtctccat gtatgcccaa 1500

gcatcaccag gaactttagg gagtaaggaa aaggcaggcc tggtgtcagc tgggctgcag 1560

atgccagctc tcccaccaac aggcccagaa ccagtttctt tcctaggttc ctttgtgaag 1620

aacttgttgg aactactaat ttatcatgat gcataaagct tgttgtcata ccctacagta 1680

ttattttcaa aacctgaatg tttttggtga cctttcatgt gccacaaaat gtaaaagcag 1740

tcatttttta aaaagtgctt gaaaaagtct agtaaagatt cttccaagca agcctcactt 1800

tctcctgttt agattgttta atctggaagg aaaaaattct ttctcaaatg acagggtttc 1860

tggtgctctg tgtttgcctg gttggctctg ggtcatctgg ggatggaggg tccctgctct 1920

tacctccagc agcatcactc ttgtctccaa agaagcagca acctcaggtg ggagaatggt 1980

tatactcaca gcattctgct tttcatgttt gaaagagggg atgggtggtg gggcatggat 2040

gtgggatttt aaaaaaatat ctaaaccata aataaagtat tactgcaatc tctttactga 2100

gctcatggaa aaactcaagt catcgaatgt tagttttgca gactggagaa gtgaggtcca 2160

gtgaacttgc ttgacttgcc ctaaatcttg ctagagagag agctggaacc agatggcagg 2220

gctcctggcc tcttacatac aaggagcatt tttcctagaa actgcaatgc agccaaattc 2280

tactggtctc aggggaaact tgttctggga gtcagcctga gcttgaatcc ctttgggttc 2340

ttcccattat cctatgccaa gcagtcatgc tgaaaccgag aaatgttttg ctttcaataa 2400

atgaaatgag cattttcaga taattatttc tgtagttgct caaaactatc atattgtttc 2460

attgaaccct actatataga acaatgactg gggagaggta ataataataa tagcaatgca 2520

tatttattgg ccattttact tgaattgtat catgtaatct agtttagagt cctgtgaggt 2580

aggttttatt atcctctcta tgaggttgaa taacttgccc aagaccacac agctaggaag 2640

tagaaagact ggtatttgaa cccatcttct ccttttcttc tccttcctcc tcctcctctc 2700

ttccaacacc tgctcccaag gaagctcatc cagtgcatga ctttagctac cacctgctcg 2760

tagtggtgac tcaaatctgc atctccaatc ctcataccta tcctgagctc aagacctttg 2820

aatatagctc cctcctgtcc atccctcctg gaaatgcagg tggcttgttc acacataatg 2880

tgaacacaaa tggagcactc tcctcacaca cccaaatgtg caccttcacc agcgtgccca 2940

gcacaggcat cccttcctgc cagctatgag cctcgaggtt agctctactc cccctcccta 3000

accctgcatg cccaaggggt ttccaagtct aatcaatgct accactaaaa tctcccatac 3060

acctgttccc tcctctccac tagcttgatc actccccatg caggccctca gttgctttat 3120

gctctcagta ggccctcctc cagtgcccac actctctccc ttctccttcc caccttcttt 3180

ctaccagagt tctaacctct ccaagccccg cttgtctttt tctttccctg gctgccatcc 3240

taactcgccc cttcccttct cagacaagct tctacatgct actcatctct ccatcaaacc 3300

accatattcg ggctttggcc atctgctctc cacagccaag tccccagtgg cctctctgct 3360

tctgacacag tgaaagccat tcagatctgt cttgttggca gcattcctca ctttgagcag 3420

cgccctccta ctaggatacc cctccttgac tacaacccca cattctctacttcctgggct 3480

cttctgtcac tggaggatga ctcccaggtg tgaatcttca tcccgcgtcc ctcactcaag 3540

cccccgatcc tcatatccag ctttatcctc atgggatgct tcaccaggat gagtcataag 3600

cacctcagac tcagggtgtc ccaaaccact catctacctg gcaagcctgc actctgcatg 3660

tgcctcattc tgaacatggc accatcacct gctgcaatgt ccagaccaca aacaccctac 3720

aatatccttg actctccttt ctccccttct ccctgtatac agactccaaa ttctattgag 3780

actattacct cctacacccc tcacatttgc ccagccttcc ccatctctgc ctctaccacc 3840

atagttcaag ctctcccatg gtcccttcct ggttacctgt tcttcttgcc tccttaagcc 3900

tctcatgaca ctggccatgt cacttgcctc cacccatcac ccgctaggct cttagctgga 3960

gtctgggccc tgctaccttc ctccccttct tccctaccct tgactccacc tccctgtgct 4020

tcagccaacc agataacttg agtttcgtga atgcatgcct cagtttacct gattaactca 4080

ttttcatctt tcaggcctca gagcaggtat caccctgtca gggccaggtg cctcttctta 4140

gctcccaaag ccccagctac tcttcatgga acatcattgg cttgggctac ggatcttccc 4200

aaattggagc tttttcacaa agggcttagg tctcactcat tctattaatc catctgtgtc 4260

tccccagggc tagcagtgcc aagtaactga caggtgatta atagatgctt gggtaagtat 4320

cacctcttta ccatgtgaca atttgtttac ctgccttgag ctcctccagg gcaggactct 4380

tgcctttgca gaatctatct ggcaggtact gttgcagaga tgtttactga agaagggaat 4440

gaattagtac caaggtgagg accccaccct tccccacggg ctccaaaagc agcttagagc 4500

ccaacaaaac ctgccccaca tttttggcgt ttctgtggat cacacgattt actcatctgt 4560

ctttcaatga gcatgacagg tggggtgggg gtggagggat tagagattga ggagctgggg 4620

agggtggtca gctcctgggg tgcagaaaca agtctgatgg gccatggtgt tctgggaatc 4680

agcactgcct cccctcaccc ctccctgcag tgttttgtag cctcaagatc agtgagggaa 4740

tcttcgggcc cccagcatgc aggaccgaag cccccgagac agctgtccct cagtcccaag 4800

gtccccattt ggaagcagcc acaggaggcc taagggacct atacccttgg tttgaggaag 4860

actgtggcga gggagagagg gagggagggc tggcagtgag ggcaagggct gggaaaactg 4920

agcacgggca cagtgcggga gcgggtgggt gcccagggca gccaggggcg cacgggttgg 4980

gaggcgccag gcggcccgcc ctccttgcac gggccggccc agcttccccg cccctggcgt 5040

ccgctccctc ccgctcgcag cttacttaac ctggcccggg cggcggaggc gctctcactt 5100

ccctggagcc gcccgcttgc ccgtcggtcg ctagctcgct cggtgcgcgt cgtcccgctc 5160

catggcgctc ttcgtgcggc tgctggctct cgccctggct ctggccctgg gccccgccgc 5220

gaccctggcg ggtcccgcca agtcgcccta ccagctggtg ctgcagcaca gcaggctccg 5280

gggccgccag cacgggtaag ccgagccgcc tggccagggg ctgcggaagg tcaggtagtc 5340

ggggctcgga gcgcaagccg ctgggggcat tgaactgggc tgggggcgca ggggacaaag 5400

cccgaactaa aaaccttgca gcatggagcg ctcggacacc agccctgcac gcggtggaag 5460

gagagaggga gggaggtgga ggaccatgga gggaaagcgg gaggccgccg ctttgtagaa 5520

gggagtgggg aagtggacca gagactttcg acgcaggcca agagcctgag acggacagcg 5580

ctttcagctt ctcctcccag ccactgcaga aagggggaaa tggcaactct ttggccataa 5640

tcaccgtggg agggtgccaa gggcaaagcc cacccagcag tacacctatt ccaacccagc 5700

caggcccccg gccagcgact ccagacaaga acctgggcca cacacggtgg cagcatctaa 5760

ggtgccccag gctcctgtgc tcctggccag gccctgcact cagacactgc tggcacccga 5820

cactgctctc tgggtacagc aagggcaatg tggcacttct tgtcctgccc gatgaagagc 5880

aggagaatgc actgggccct cacacacact gttcaaatgg ggaaactgag tcctgagtgg 5940

ttccactttc ccacagtcct gaagtgtgca ctggagccag gattggagtc tgtcttaaag 6000

taatagctgg gtttgtaaat gtaggacact atcattgcag gaattccttt gagaccctga 6060

agatgtgttg gctttaggag acaaactcaa gcagaaggtc tggtctgata gtggccctaa 6120

tactgaccca ggcagaggca ggcaacattt ctacctcaaa aaccaggcca tacctgcgtc 6180

acaaataccc aggctttgct gcagcttcca gcctacctgg ttgcaccaac ttctttttca 6240

taactaggta aaactatata tgagtagaat cttgtagtga ctcctcagag gaagcctaaa 6300

taccatcggg gtctggcgtt cacacccaca agcaatgccc aaacctccaa gagactgggc 6360

agatctgtgc tcaaatcaaa actcattgtt gggggtgata gagttgactt cacaggccct 6420

gaaagtcttg gctccttgca ctaggagtgc tctgggtacg ggtacaggct gccccttgta 6480

gggcatagtt gctcttgttt cctctacttg tggctttatg gtctaggcct ttcaggagtt 6540

tggggctctg gcggagaggg cctgctggga gcacatctgg ccaccctgca gagtgaaatc 6600

aaaccaggcc tggctgcaac ctcaacaccc tcctggaaag aggagaatac tggggatatc 6660

ctggggtctt tctggaagtg ggagaatcag ctttgacttg ggcagtgtgc agaatagagt 6720

gaggggggat gtcagaaaga tgagagggat atgaggcctc aacatcaaaa tgcaagcacc 6780

tggcattttt attatctctg cccacctctc cgttggtctc tctgcctttc ctgccaatga 6840

attgtgttat gtttgggtgc ctcaatttgc ctaggagggt tctatttctt ctgtatcttc 6900

gccactaagt caggagaaga tccttatagc atgccctgca acagtgtcac ctgtaagggc 6960

atctctctgc acagccacag tgaaggatcc tcaaaggtat tgagggcttt ccatcaagag 7020

ccatctttac agcaaacctc tttcccttca gagcccagaa gagtgctgac cagctggaaa 7080

acagggtttt tttcttaaat gcagatgctc ttgattatga gttccagata ttagatcaac 7140

ttccccacca tacccctgca ggcaaagcct cttaattagc ttcctgcagc acagctggaa 7200

aggcctattg taatctgtga tgggcagagt aatctaagaa gtcacaggag cacccctgtc 7260

ccagtagaat ctggatgcgc aggcacatga accatggcaa aatggttgca ggcacagttg 7320

tatttactct gatctaactg tccctgttaa tgccacaggg ctgcctggcc tggcacacag 7380

ggctgtggcg ccttgtgcaa atggataacg ttgttctagc tccagccttt cattcaaagt 7440

gaaaactgtt agaaagggaa ggaaaacttt gctattttaa ggaattgtag cgtgctgcct 7500

gatatgaagg aagaaataac agctgtgcct tgcttgtgcg cagcactcga ttgccgcttt 7560

tgctttcgac ctcaccacaa cacagtgaga tctactgttc atgttcccat tttacaggag 7620

gtgaaactgc agcttagtga ggtagagagt gacttagttc agacacagaa tgctgttggg 7680

agagtaataa ctatgatatg gtctcttgac tcccagctat atctgtgttg ctatagggaa 7740

ggggaaaaat aatactgaaa gagaagtaaa aatacaatca cacttccaaa catcaaccac 7800

caaaaactga actgaatttc ctgaagcact tggttttcaa atctaagctg aacatcaatg 7860

ctgttattct tgaggcccag aagcaacttg ctcatttcaa ttaagcttca gcatgaactt 7920

cctatgtaca cagcccaccc acactccccg atgtgagaag gagagggtca cagccgcccc 7980

cagcctctgc tgctgccaca aggacagcag cagtggaaac attcagcaaa ggaatgttgg 8040

agccacatcc acaagagact cactgaagat tcgccaaacg cctacggaaa gtggcaggga 8100

attcattgac agtaattgtt tcctgcttga tcagattgaa gagcttctgg gattctgtaa 8160

caataaatag gaccgggggc tggagtatgg ccagcaagga ctcttcaggg gttattcagg 8220

gactgtctaa cctgtgaatc ctaggcagca aacagaaacc aggtattcag aaatctggag 8280

gatttggtca ggcccagcta ggactaggga ggcatgggcc tctgctggct gtggtccctt 8340

ctccagcctt cacttctctt gtccctagat ccttacatgg attcattaat gctcattgtc 8400

cctcctgggc ccactcactt tcacctgttg aacaaaaaac tggccaagag gtgacagtca 8460

tatcaccgca gaagagacag ggcagagaaa tgaaggggca gaatggactc ccacccaaaa 8520

gcctgactct gaatatttga gaattgttca agttcctgca gaggaatcat gatggggaca 8580

gtaggtgtag tttttactgc aatattggtg tcttcttaac aaatacgctg cacatcaagt 8640

gatgtctgtg gatggcattc ttaaagtaac agggaaattg atgttaaaga aatacttcat 8700

cctttgggtg atacctgaag ttctctgagc ttggaggtct tgtgaaagcc ctcagtattg 8760

tttgttttat ttgctttcct ctgacttgtg attcagtcag atgcatgcct gcctctggct 8820

caggaagatc aaccctctcc tgactgacca cgcctctcct gactgaccac gtagcacagc 8880

agcttcctttccctaggggc tcctaatgaa gctttcacaa tcacctggcc tgagcacagt 8940

ttgggtcagg acttggtata cttgaaaaaa acatgcaaaa ccaaaatcct gtggttctgg 9000

aaaaggcttc ttagcagaac ccccagacat ttacactctg ctttttcaca gggtccctga 9060

ggattctttg gatctgggta gtttggggag cagtattttc aacaagttca tttcgtgctc 9120

cttctacacc ctgcctggat gctaggcccc atctagaatg tgaacaacag aacaaggcag 9180

aacacttgtc ctcaaggttc tgttgagtgt tagatgcaga gaagagacac cccccacctc 9240

cccgcatcac ttacaggaat tctgtttgga acccaacatc aaataaggac cgtatccact 9300

gtcagaggat gggaagcagc atgtcatctg ggacattgga gaaaggctcc tgggggaagt 9360

gggacttgag ctgtgatcta agtaatgaac aactgagagt taaatgggag agcatcccct 9420

atcagggtcc tgagagcaac cagccatggt ttaaaccagc tataaagcct cgggtttata 9480

ggatagacag taacaatggc ttgtctttgg gagccaagca gctggtccag gcatgcagag 9540

catgtctgta tggagagctg cctgagagat gcttttgttt acacttatca attgcccatg 9600

tcaaagaagg atatgtacat gaagttacat cagtatgtaa gagagatttt aacaattttt 9660

gcaggggaag ctttcatggg ggctgatggg aatctaggta aacagaacca aagtctaaac 9720

ccaagatatc cccagtacca agactgaaat gactctctcc tctatctcta gaaagttcca 9780

gtgacccaag gaggcaaaca cgatgggagt cattaaagtg gggtggacgt gctgatcatc 9840

ttcctaattc tgctgctttt gttttcagcc ccaacgtgtg tgctgtgcag aaggttattg 9900

gcactaatag gaagtacttc accaactgca agcagtggta ccaaaggaaa atctgtggca 9960

aatcaacgtg agtatctgta accagccagg agaccaagct gtatgcacgc tggctgcagt 10020

tccccagggc ctgggccagc cttctagaag gtcaggttgc ctaaaaagcc atgaagatgc 10080

atgtgcgaac atgtctggga cctgcgtgct agggagtggc atttttagga agctggccaa 10140

ttttgttttg catttttaag gctgctgaca agacttggag acatttttca gggctggttt 10200

gggtttgcaa gaaacatgaa acactgcgtg tgtgtgtgtg tgtgtgtgtt tctcaatcct 10260

cataaaataa tacagatatg cagtggagaa gccaccagca tgtgactctg gaaaagaaag 10320

cccattggtg aatctgtact aaagaatgcc atccctatct tacagtccta aggtaaacac 10380

cccaaaaaga cttagagcac taaacatatg cagattatga gacagcatag catataatat 10440

ttgcacagac ttcctcattc aaaccctagc tctacctggg ccagtcgatt catctttaga 10500

accctccatt gctttacctg aaaagttcgt ataacaaaag gacccacctt atggggttgt 10560

tacaaggatt gaatgaaata atgtacataa gagactgaat atggtgccca gcatatatca 10620

gtgctcaata aatgctagct actattatta ttatcaccct agatttgcaa atctagacca 10680

cacaagcaga agtaagagtg ccaacggggt gtggaccagt gtggttacaa tagggcttgt 10740

tgatgtctgt ttcagcaagg agggaggcag cttttacccc actgcccagc tccctggtgg 10800

aatcaggtgc atgttctaac aattctgggg aaacctaatc tgttttggca ctgtcaacag 10860

atctcaaagc tggctgtctc ctatagctag gaagatgtgt atgacaaatc tcctgagcca 10920

cttgtgaagg cctgaccttc ctcctgtctc catacataat gggatgatta agaaactcta 10980

agccactctc ttaagcactt ttcaatgtta gggattttta agtttattgt tgtgacattg 11040

cttttgagca gacatctcct ccaatttaat agccaactga aagaagagaa aatgctcttt 11100

ccttaaactg tatgtggaaa taaatattcc aatgtgtgac cctgattatg ttaggcaatt 11160

agcaatccta atatgaattg agggaagttg ggattcatgg cacagctggg gagataccag 11220

cagtccctgg gagcctgtcc agggcaggtc catggcagct tgctccatgc ctgattgaca 11280

gcccagcctg caagctaaaa gttgagtgag ctaggaggac acactgccaa gattcagcta 11340

acagacaccc agcgatattc ttgctgctat gaacaaaagg agactatgca aattatacac 11400

cacccattct tccaggatgc ctgacttaaa aaataagaaa aaagatgggc cgggcacagt 11460

ggctcacgcc tgtaatccca acactttggg aggccgaggt gggcggatca caaggtcagg 11520

agacagagac catcctggct aacatggtga aaccccgtct ctactaaaaa aatacaaaaa 11580

tattagcggg cgtggtggcg ggcacctgta gtcccagcta ctcgggaggc tgaggcagga 11640

gaatggcgtg aacctgggag gcggagcttg cagtgagcca agatcgtgcc actgcagtcc 11700

agcctgggtg acagagtgag acaccgtctc aaaaaaaaaa aaaaaaaaag aaaagaaaac 11760

ctttagtact gattgatttt ttcccatgtg tgtatattat ctactcaaat taacaattaa 11820

ttacttaatt aaacacaaag ccaggcctca cctaattgct tcttggaagg tgaccagagt 11880

gctagtgcca agcaaacaac tcttctatat ctcaagagcc ctgggcttca gagggccatc 11940

ttttttgtta attcaagttt ctctgaaaat ggagacccgt ttatgatgac aagctggcta 12000

cagggtagca tctgccacac tgtttcgggg gtgccgctgg gctgaagcat ttgcccagct 12060

agttaacaat agctcgataa cattccctat cagtgtccag gctgagaata ctgtcagtga 12120

tgagtcgcct tggctcttgt acctgtatct ttgtgtgcca ggacaaggca caagcaacag 12180

agctgtgtgt tgccaaaatg ttcctgatga gcaggtcaac ccctcggggg caggtttgga 12240

tatgataatg tggtgatgtg gtggcgcagc tcccttaccc agtgagcaca aggggagtcc 12300

tctaggaaaa ggaagaaatg tctggatgag gtggggagat ggggttcaga gtggactcag 12360

gcaaagcccg atgcccagtc ccagctgttg gcctagtctc acaaagccag aaggatatga 12420

catttacatt caactcttga atttgtggcc actgctttgg gcaacttcaa agagagaaaa 12480

tgaagataga aaaatattat ttgatataaa acttctagga caagagaggc ccttcctgga 12540

acattacatg tagtattagg aaggtggagc tgccctggaa aagatccaga gaactcagag 12600

agaggaagag gtggaaccca tctctgttct tgtagagagc tcagtaagag tggcttggca 12660

gggctcctgt gtacctgaga ccaagaccag tgaggaggct actgtctgac caccatacgg 12720

tcagaattca gtgccatggg tggtcaggtg ggaaggggag aggactgtgc tggctggagt 12780

tgatgttatc ctggggaaag taggtcccta gatgccttta gttgagtgag gagcagactg 12840

ggaaatggga gcacagtagt ggttggggca aaaaggactg tctctgcatg aggtccatag 12900

gcagttggaa ttttctcagc aagactccag agaaggaggc tggagcagag gtgtatgttg 12960

ggatgaaaag gagtaaagta tcatggggga ggaggcagct caggttgtca agggtcaaga 13020

aaccagaagg agaatttcac cttggaagca gacaacgggt accaagcata caggggaata 13080

ctttgtggtg agaggtcaca cagagataca ggagccgacc tggtgagaca ggagcctgga 13140

gccacctgcc tgcttttgtg aggccccaga ctccactgct atcatcaggt gaagctctgt 13200

tgcctgcaca caaaagcttt tctgcattta caaagagaga agggcctgag tttctggtgc 13260

aatgcgtcaa gctgacatat ggactttatt acaggaagtg gttaccagtg ggtccctatt 13320

tagtggctgt tattgtgaat tttattgttc ggaaattcac tttagcattt atttcagatc 13380

ctaaatagca ccggagtgat acaatggcta atcaaacaaa gagggctgtg gggagcagac 13440

agtcagcatc cccctctgtg atttcaggcc ctggtttgat tagtagccat aaaatttttt 13500

acgtgtggca ctttgagcaa aggtgcagga aattgtggtc aggaagcctg gctgcctctc 13560

gacaggcttc ctttgtgcta gccccaggga gaggaggcct atttaacagc caagtccaag 13620

ttgacatcat gggactggaa tagtcatagc aggagctcag acatcataaa cgtggcatag 13680

ggagggctgg tggaggagct agcgggtatg ggtggcagct attcattcca aaagtcttga 13740

aattgtttca cgagcaacac atttcacaag tgcgaagccc ttctctggag ccaagatgag 13800

ctggcagagc actcctgttt ctctagtagc aagtgttcct ttgcccaggg gcaaaaatat 13860

taatactcct tcagcactgc attaatgctt aaagatttaa cttttaaaga gatcagctgg 13920

tgcatggtcg agcttttcca tcagctggca gggctttttc agtaggtgtc cttctgggca 13980

gggcactggg gacagctgac gtgaaggtga agaagagctg tcgttttcct cccttatatc 14040

ccacaacctt ggtcccaaga ggaaaaaaaa gaagatggtg agaagtcatc caagcagacc 14100

ccagacccat actagtgcct cctttcctgt ttcatatccc tgtgcagcca gctgggatct 14160

cttgaataat ctgctctggg ggcactgaga ttggacatac accaaacagc ggagatcgac 14220

caaacgcctc tgttgggcag tgtttcctga gggttctgtc ccattctgta aactaggagg 14280

ctgactagct gacaaggaat tttattctgt tgggtattta catgaaccta tgtgccacct 14340

ggggtaagac cctgtggtag gtagaaacat gacttcccaa aaatgtccac atcctaatct 14400

ctaattctgt aaatatattc ccttactgga aaaagagact ttgcaggtgt gattaaatta 14460

aggatcataa gagggagaga ttatccagga ttatttgatg agtctaatat aatcatcagg 14520

gtacttaaaa gagggaggca ggctgtgcct ggtggttcac gcctttaatc ccagcacttt 14580

gggagactga ggcgagcggg tcacgaggac aggagttgga gaccagcctg accaacatgg 14640

tgaaactccc cctctagtaa aaaaaaaaat acaaaaatta gccaggcatg gtggtacaca 14700

cctgtaatcc cagctactca ggaggctgag gcgggagaat tgcttgaacc caggaggcag 14760

aggttgtggt gagctgagat cgcaccactg ccctccagcc tgggcaacag agcaagactc 14820

catctcaaaa aaaaaaaaag agggaggcag tgggatcaga gtcagagaag gcaacgtgat 14880

gatgaaagct gacatttgag tgatgcaacc acaagccaag gaatgcaggc agcttctcaa 14940

agctggaaag gacgagcaat ggattcttcc ctacagcctc tgtgaggaat gcagcctttg 15000

attttaaccc cataaggccg atttctgact ctagcctctg gaattgtaag ataatttgca 15060

tgatctcaag ccactaaatt tgtggtaatt tgtcacagaa agcaatggga agccaacaca 15120

ggccttattt gttgacttat agatgcattt ttctttattt caatgtactt ttatcaatgg 15180

tctcatgtag ggtattgctt tcaatgaaga tattaacata gtttcaactt taaggtttat 15240

atctggagtt tctttagaag cttcacaact gaccacttag taaacagtaa gcatctgtta 15300

agtgcttctc atatgtaagt tcattcaatt ctcacaatca cactataaga taaatatgat 15360

tattagccca tttacagatg aggagacagg ctcaaaagac ttttatgcaa cctggtcaaa 15420

gtcattcact ggtaagctga ggaggtctgt ccacttcctt ttgctgcccc cagggggtat 15480

caagcctggc agttagtgtc agcgacttag gaggtgaaca agtgagcagg cctgtaggac 15540

ctggctaaac tgccccaggt ctctgtctac agcctcaaac ctgtggctgt gggtcccaga 15600

gacaaggcct cctcagcatc agagaaggat gcctttgtct cagggtcatc aaccttctcc 15660

aggttgctca ccccctgctg taaaggggat ccccaagacc gctcatcaga caaggagctt 15720

gggaactgag gagacacagt cagcctccag gagtgcccaa aatgccctca catgctgcat 15780

acagattgcc acaaataaag tacatccaca ttctgaagac tctgtcctca tcaccaacca 15840

ggctggcccc tggtgagggc tgtagtggtt gaggcctttg ttggtagaca gtaggttaaa 15900

gcaagccatg attttctatt gggaggcttc agaatcagct cagctgtgtt tccaagacca 15960

ggagggcaga aagcaaacca tcccaggcaa gcagtccatg ggccatgtca gatgtctaga 16020

cgttatgggt ctgtgtttgc tctgccattc ctctcggaaa ctatgatgcc ctgtatggtt 16080

taccttcagt cacaggtgac tggcctacag ggccattcct tgttccaacg acttctcgag 16140

tataattaat ccccaggcat ttacggccag agcagccggc caaatccgtg aagtgcagtg 16200

gttgttttaa attatattaa cttcttggaa acttatttta gggagagaaa actcagtact 16260

tctctctatc caatcttgag taaaaatgtt agaagggact ggtggagagc ctcccagaca 16320

tccctacaca tagactttgg gttgacatta tctctttgca ccttccttga aactttcttc 16380

taaattaggt gccttcccta atttaggcac cttcccagta ctagtctgtg acctgttagg 16440

aaccaggcca cacagcagga gttgagtggc agggagtgag cattattgcc tgagctccgc 16500

ctcctgtcag atcagcagtg gcattagatt ctcatagcag tccgaatact attgtgaact 16560

gtgcgtgtaa gggatctagc ttgtgcattc cttatgagaa tctaatgccc gatggtctga 16620

gatggaagag tttcatacca aaaccacccc ttccccctgc caccatctgg ggaaatattg 16680

tctaccacga aactgatccc tggtgccaaa aaggttgggg accgctgtcc taagggatct 16740

gctttttctg acctgaggtt tttctttatt agactgtatc tggctgagga gaagcctgaa 16800

gcctttaatc ggaacagctt tggctgatga gattagattc agaaaccaac agattggtct 16860

tttctatgca gggaagccta ggaactgggg ggctatggct gggaagcccc ctattgtttc 16920

catcctttcc tatgttcatc ctggaggaat ggcatcagac ccatgcctct gtgattgctc 16980

ccagcccatc caaccacagc atctatgttc tgcctgggac cagggccagg gagcatggca 17040

cactgagctg agtataagga gagtggagca ggccactgcc agcccagaaa attttggtca 17100

aagttgcctg aaatcttctc agccttcgat tcacagctgc tctctgctgc tctggggcca 17160

tgcagaccag ttcagaaaag agttaatttg ttggggcagt tggaggcagg tggactgcca 17220

gctttgacac cttcccagcc cacaggctgc tgcactgggg ctgaaggcgt ggctaacccc 17280

tgcacaccta gagagtgaca gagatgccag actgggcagc aggaaggcaa gaggattaag 17340

agagagcttc ctggctgaaa gccacactcg gttaaccagg aaaaagccct tggcacgaga 17400

agactcagtg gcctgaggga ctgagccttg gttgttgggc atgtgctgca taagccatcc 17460

atgtgtgaca gtagagtgta gtccagccac tgtgggacat gggtgctgaa agaccacatg 17520

gagaggaaca gtgagtgctg acaagggcta gccttgatca ctttggagac accccctgtg 17580

tcttctagat gtcagacttt ccaaatctgt ctgctatcct ccaaacgtgc attttcaaga 17640

gcaatggaaa aaggattgga cttgatggaa tgcagcaaga gtcctaggtc tgttactacc 17700

tacctatgac cttaagaaac tccttcaccc ctcagaaccc ttacagcttt ctttctgatt 17760

ctatcctgag ttactctact ccaagctgag acttttctgc ttagatctat cccttcctcc 17820

taaaccccca acctccattt ctcctggtgt ctttctttac acacccctca gcatacacac 17880

acacctagcc acaggaacca atgagttaat atttgaggag ttggttttct tttgtcctca 17940

atgagatcct ggtgaggcca cttgagctgt tcagctccct tgcggtattt tggggatgga 18000

actcagaagc caacaatata gaaaaagagt ctttggccag ctttcccagg ggctccatgc 18060

catagagagt actgcacccg tgtgcacagg gggccctgac atgaggactt tgaggataac 18120

actattcctc caactctgct tcagcatctc catggatttt cacacagaca ctttaggaaa 18180

gaaactaagt ttggggggac ttgacctaat cccacatcac agccccagta atacagccct 18240

ggaatttatc acagaaagcc tagaatccca tgcatatccc atgcatatgc atccctagtc 18300

ctatgggttc aaggcttgga gctctccctg gatttagctg ggaaaagttg gcagacagtt 18360

cttctctgtc ttctagaaat atggactaga atcgtgagtg tgagattgca agtaactttt 18420

aaaatcatct agtttaactt caccccattt catagaccaa gaaactgaga ccagagagag 18480

aaatggactt tcaagttcac cctgctagtt actgatggat cacaagtcaa atctcctgat 18540

tctagcactg tttctcttac accacaccac ctttgaaagt gtgtcaatca aatcttactt 18600

tagttgcaga ggatgacttt agtttctgaa gataaaattg tgagtcaatc aagatgagtc 18660

ccaagacaat agcctgttta gcccttataa gttcagggat gaaaggttag aaagaaacag 18720

gatggaagga ggactggaga aaaaaacaaa agaggaagga aggaggagga agcaaacagg 18780

aaaaaaaaag aatgtgcata gcttgtcact cctcagtcat ttcctgggag cccatttcta 18840

gcaaagtgac agctgcaact ccctggccac ctgagcatct tagctgatct gtctctgaaa 18900

caccccctgg agaacagatg aatcaggctt catcttcgct taactaagtc ttccctgaga 18960

cgactccatt taaatgaaca agagcaggat ttcctgggca cactgagagc accttccaga 19020

ggcccctcca gagccctaaa gcctgtattt cttccagtcg gcctgtttct ttcctggtga 19080

tgtcattaaa cgccctttga gagtcccaca gtgagcagtt ctgcggtaaa acccgctgca 19140

attaaagtct gagtcctttc ctgtctcaaa gggcatattc atatagaaga aaggaaaagg 19200

aaggactggc tgtttgcatt tggttccagg cctgttgagt agaggtcgtg ctcactccac 19260

cgaaggtaca gggtagcctt cagcagaacc tggggatttg gttttaagca agtctttctt 19320

aggtgtgggc tttcagaaca cttccttcct tgcaatatta tttgaaattc tcagtgtttt 19380

agccgtcccc agaatattgg ttcgttaaag ctgtgtattt cagatctcca gacagtggtc 19440

actgtttgta tattttcaat ttcaaaccag aaaacaaaag ttcttattga ttactttttt 19500

tatttaaaaa ataaaaagta agtatcttcg taagaggagc tttgttttaa ttttaaagtt 19560

taaaatttga ttgtgaagac agagaaaaac ttgatgattg tagatatatt cccctctttg 19620

gctattcaat cagagaacta gaaaatcatg agagatttaa tgaccactgc ctgatacaca 19680

tatgtgtttt acagatgagg aaactgagac ccagagagat gatgaaattg gctgaggatg 19740

gcccagctgg tcagtgaaag actcagagcc agagctggtg cagggctctt tctattcctt 19800

cctgttccct ttcaggaaca ctcaccatcg gctttcctgt gaataatgtt gagataaaat 19860

ccttggtgca ttatgttttc tagtcacaac attgactagg ctgccagagt cctctgttct 19920

cccagttggt tggctgtagg tgttggcagc cgccaggagc attctacaga acagaggagg 19980

agtgagactc tccttgctca ggaaaggcag acctatgact tagcaaataa ctcctaagag 20040

gagagtgttt cacccaccat tcctcttcct tggctgtgga ggcaacttag tggagagggg 20100

ccagatgacc tgtgaggaac agtgaagccc tgcctaacac aatgtatggt tgtcttgtta 20160

cagagtcatc agctacgagt gctgtcctgg atatgaaaag gtccctgggg agaagggctg 20220

tccagcaggt gaatgaatcc tccgggcctt gcctgttggt gtgggtggaa gggaatggtg 20280

ggagagagga gtacccacat aaaaggcagc agagtgtgaa tgggggcagt ggcacaagga 20340

catggcattc tccccacgtg cccactggcc ccaggctcta tgcgaggggc tgaggaatgg 20400

aagctggaaa cagcgcattt cctgagctgc tcctcctggc ctccttacca cactggtgga 20460

gtagactcca actgtggcct gtccatgccc ttcccagcag gcacaggctc aggctcaggc 20520

tcttggcctc tgcctctggc tgggagtgat tctaaacaca tccagcaggg tcagcctgat 20580

agcccatcag tttccgatca gctctgctag agagccgatg ggatgtggga ggagggggtc 20640

actggtgggc tggcaacccc aagccatccc catctccctc tgtgtctaaa cttggccctt 20700

tggagttcgg tagggagaag agccataggc caggtgggct cacccagagt cagcagagag 20760

tcccacaaat ggttgcactg ggcgaaagac agcatggcac ctgtgaattt tattagagct 20820

tttcttttag tgctacacac aagtgactgt acaggggagt tagtattttg ttttaatttt 20880

gaaatagagt catcttttgg tatctgcggg ggattgattc taggacccat tctaggatgc 20940

catatcctca gatgttcaag tccctgatat aaagtggtat agtatttgca tgtaatctat 21000

gcatattctt ccatgtactt taaatcatct caagattact tataatacca aatataatgt 21060

aaatcctatg taagtagttg ttataccctc ttttaaattt ttgtattatc ttttattgta 21120

tttcaaaaaa tatttttggt ccatgtttag ttgaatctgt gggtgaagaa cccacagata 21180

cgaagggcca actgtattgg ctattttttt agttaagaat gtgagactga ggccaggcgc 21240

agtggctcat gcctttgatt ccagcacttt gggaggccaa gaggggacga tcacctgagc 21300

caagaattcg agaccagcag cccgtgcaac atagtgagac cttgtctctt aaagattgtg 21360

agactgggct gggcacggtg gctcacgcct gtaatcctag cactttggga ggccaaggca 21420

ggtggatcaa ctgaggtcag gagtttgaga tcagcctggc taacatagtg aaactctgtc 21480

tctactaaaa atacaaaaaa attagctggg tgtggtggtg ggcgcctata atcccagcta 21540

ctcaggaggc tgaggcagga gaatcgcttg tatccaggag gcggaggttg cagtgagctg 21600

agatagggcc gttgcactcc agcctgggca agaagagcaa aactccatct caaaaataaa 21660

taaataaata aataaataaa tcatgagact gagacataac aggaaggagg gcaatttggt 21720

tggttccaag gttcctagag tatgtgatgg gagaggttgg tgcgggtggg gccatggagg 21780

tactgactca agtggaggga caggtgggga aatgggatgg gaaaagaaga ttgaccttag 21840

aaggggagct caacctctga accctaattt cagacccttc aaaatgaata ttaagctcat 21900

tttggtctaa gaaacaaaaa acaaatgaac atgaaactca ttttggtctt ataaggtctg 21960

agaaacccct tctaaacttc aagctgcttt aagaaataac attttattac ctgcaaatac 22020

acacagtact ttggagattt ataatagtct cttattctaa tagaagccat tagggaacca 22080

gtttcaataa acaggtaaat ctgtaagact agtttgtaat taggatatct gtttccagtg 22140

tccattcctg cctctgttat ctaaatgtct gggaacaaga gctgtgctct gctgtgttta 22200

aaatgattaa aaatcaccaa ttagttgagt tcacgtagac aggcatttga cttattgagt 22260

tgttttaaga agactataac aagccttaag ccccccagaa acagcctgtc tttgggcttt 22320

cccacatgcc tcctcgtcct ctccacctgt agatgtaccg tgctctctgt cagagaaggg 22380

agggtgtggt tgggctggac ccccagaggc catccctcct tctgtcttct gctcctgcag 22440

ccctaccact ctcaaacctt tacgagaccc tgggagtcgt tggatccacc accactcagc 22500

tgtacacgga ccgcacggag aagctgaggc ctgagatgga ggggcccggc agcttcacca 22560

tcttcgcccc tagcaacgag gcctgggcct ccttgccagc tgtgagatga cctccgtctg 22620

cccgggggac tcttatgggg aactgcctta cttccccgag gggtgggcat gatgaatggg 22680

agtctgcagt catttcctac tgtttcagga agctttctcc ttaacccctt agaaaaggct 22740

gtggaacttg agctaaaata tgtcttacca ggttgcgtct aatgcccccc gttccctact 22800

gggcagaaag acttgggtgc ttcctgagga gggatccttg gcagaagaga ggcctgggct 22860

cacgagggct gagaacatgt ttcccagagt tgcaaggacc catctcttaa acacagagtc 22920

tgcagcccct aactgacacc ctgtccttcc tcctaggaag tgctggactc cctggtcagc 22980

aatgtcaaca ttgagctgct caatgccctc cgctaccata tggtgggcag gcgagtcctg 23040

actgatgagc tgaaacacgg catgaccctc acctctatgt accagaattc caacatccag 23100

atccaccact atcctaatgg ggtaggggat ccccagccat actgcatggc ccttggtgca 23160

taatgaaccc atttctgttc catgtgtggg ctggtttctg gggtttaagc tgtagacaac 23220

ccaccctctt tgtgcctgct tctccttggg ccctctattc cacagcttgt ggaacccaca 23280

ttttgctact gtgtttgaaa acactgtttt ctcctcccgg ggctttggga ctatgcctct 23340

gttgtgttga ctgctcatcc ttgctgcttc tctgggcaga ttgtaactgt gaactgtgcc 23400

cggctgctga aagccgacca ccatgcaacc aacggggtgg tgcacctcat cgataaggtc 23460

atctccacca tcaccaacaa catccagcag atcattgaga tcgaggacac ctttgagacc 23520

cttcgggtaa gggactgccc tgggtggagg cccaggcttg ggacacattg cctcccaaga 23580

ggggcctagc aggaactctt ctgcaggaga ggtagaggat ggctcctgta ggggaacata 23640

gagcaggttc ccctgaatgc ccttgaacat ggagaattca ttgaccagac attcagcttg 23700

acctaacctg tgaaattctc catcttcttt ataaagtgtt cccttccttg cctcccctgg 23760

aaaggtcagt ggtgtgtggc tgcagcagca cagtgtcctc tgagccctgg acctgcactg 23820

tggcttccag aggtggcagt tcccacatgg ggtactagaa taaatggcct atcaggctgt 23880

gtgtgctttg ggatcacatg tccccaccct aggaccctgg ttccaaccat acgcatgttc 23940

tcttggagcc cagaacagca gagaagccac cagtgtggac acagaagtca agggtctgat 24000

ttccagcctg gcttctgact gctctggggc cgcaggaata cggttccttc ccccatgccc 24060

agcaggcatt tgtcttacaa ctggagggga aggcatgttc ctcttggcaa ggactgctca 24120

ggaggaagtg gaggcaggct gccctgtcag ggtttttgcc ttgattcaag gagaacttcc 24180

taaccacaaa ggatacaagt gggagtgagg cggaccctcc ctagagatct ccaacacaga 24240

gagacaaaca cgctggggct ggctggcact gacaggcctc gcaggtgtgg atggctgtta 24300

gctgggagct tcgctgtcta agctcctctc ccatgctttt cttctgggtt gctcgaagga 24360

cgggggtctg caagaaaatg atgttcccac atagttggca gcacgtgaac agcaattgat 24420

ccctttgcat cacctcctct tactgtttag atttggtaaa tatttcttcc ttccctcttc 24480

tgaccctcca ttttgccgat ctttccttct tataacacat acttactagg tacctgctac 24540

ttcccgggtg ggcctatgtg ccaggagtat agaggtgaac aaggaaggca aagttctatt 24600

ctcagtagag ctaatactct atctggagag agacaacaaa caaatcaaca aggtagccag 24660

gggctgtgat aatttatgtc aagtgggcag gtaaatcggg agtgacagta gtgcagggag 24720

gattggaaag tcagggagtt ctctctggag gaggtggctt ttgatctgca gcctaaagga 24780

tgagaatggg tccattatac aaaatgctgg ggcaagagca cacccagtag aggggagagt 24840

aatagcaaag gctcagggca ggaagggcaa gggagaggcc agtgggtgag gtcacatgtg 24900

aagggcatac aatgggcaaa gacaaggcca gagtggccag gcccaatcct ccaggacttg 24960

cagacctggg aaagagtgca tctccatcct gggagcagca ggaaaccact caggccttta 25020

gaagatcctt ctggcagctg tgtagagaat gggtggtgtg atccttccat gcatgggctc 25080

atgtacgtga ttaccagtaa ctgtcgagtg acagtgtgag gagggctgca agccatgagt 25140

gtaggcacag cagacagact cacctttgtc tggcggtgag atggggtggg aagtgtgcca 25200

agttgacctc ccaaagaaat gatattttag tggaagaatg aatagaatca gagaagcaaa 25260

gtaagaggga agagcagaga ggacagcagg gacaaggact tgggggcagg aagaggaaag 25320

gcaggttaag gacatgaaag atggccaggc tggctggagc tcaggcccag caaggccccc 25380

tgggggccat ggtcatgggt gagcttgggt ttggcttctg ttttcgtctt gggcttctgt 25440

gaaagcctcg agcccttgcg gggaaccagt gaagctgtgt gtgcatcttc tgtggggagt 25500

gccagagtct tcagggagca ctccatcttc tctcctcccc acaggctgct gtggctgcat 25560

cagggctcaa cacgatgctt gaaggtaacg gccagtacac gcttttggcc ccgaccaatg 25620

aggccttcga gaagatccct agtgagactt tgaaccgtat cctgggcgac ccagaagccc 25680

tgagaggtga gcatcctttg gctcctgctg ctgcctcatt tgtgcagcta gattgagccc 25740

aagacctgct ctggtccaag atgaacatac cacctgccat gaggtgaccc tcaggatatc 25800

cactgcagcc atgggctggg gtcatcctgt cctgttgctt cagctaaccg tgtctctagc 25860

agccacacta ctctgagggc tgactacaga atccagcagc ttttgtctgg gagagctgga 25920

ctgaagagag gcatagctgg agacccatag ctggccctgg ccagaaacag ggagagtgaa 25980

aggctggaat agccaaggcc agagcaaggc taataggtag agcaacagct tacaggtgtg 26040

ggggtggcag atactggcac ccttgaaatg gattcctcat gcccacgctt cactattctt 26100

ctctgtggct aggggattta tggataaacc aaaattacag ttaaaaacca gccataggcc 26160

aggcacagtg actcacgcct ttaatatcag cactttggga ggacaaggtg ggcggatcac 26220

ctgagatctg gaatttgaga ccagcctggc caacatggcg aaaccccatc tctactaaaa 26280

atacaaaaat tagctgggca tggtggtggg cacctgtaat cccagttact caggggctga 26340

ggcaggagaa ccacttgaac ccaggaggtg gaggttgcag tgagccaagc ttgcaccact 26400

gcactccagc ctgggtgaca cagcgacact ccgtctcaag aaaaaaaaaa aaaaaaacag 26460

ttatagtagt caacttttga ctctccattt cagatttcgt catgccctcc tcaatgagct 26520

gctaagttag gcagtgcatt gattattgct gcaggagagg gaaggaagga gctaacgtgt 26580

tttcacatgt tttccttttg gagatgagaa aggaggactc tgccttcccc ctaccctgcc 26640

cctttctact ccaggacctc tgaaaggcca tgagcacaaa gctgctgcct gagtcccctg 26700

aaatgcaggg tacgccccag gtctctgatg taccccacca cacttttcct ctcaaacata 26760

ttccaggatc acttgatttc ttttgaatct atttaaaccc accgtgtcaa tgtgctatat 26820

aaaatgtcta atgcatttca gacaccctat acatctatac atttaaagtg ttctccttct 26880

atctgtgcag ggatgggaaa gggcatattt ctgaaagcac agatgggaag acgggatttg 26940

ttccgtgtcc aggtgattat ggtacctcta tgcgcctggc cggcactggg gacagaggcc 27000

atgaaaatga atacagcaca gcctttgcct ccaagaaact taagacctag tagaaatggc 27060

aggctttaaa acaggttgtt gggatctgat ttggtgagtg caatgacaga gatactcaca 27120

gcacaaaatg gggaatgagg gcgggcattg ggacacacat agccttaagg ggcccaaagg 27180

cttttagaac tgtattccct attaaaacat gatttgcaca gagcacattc tttgctttgg 27240

agacctcaga actccttact ataggccggg catggttata atcccagcac tttgggaagc 27300

caaggcgggc agatcacttg aggctgagag ttcaagacca gcctggccaa catggtaaaa 27360

ccccgtctct actaaaaata caaaaattag ctgggtgtgg tggtggccac ctgtaatccc 27420

agctactcag gaggctgagg taggagaatc acttgaacct gggaggcaga agttgcaata 27480

agcccagatc atgccactgc actccagcct gggcaacaaa gctagactct ctcaaaagaa 27540

aaaaacaaaa caaaacaaaa caaaacaaaa aaaactcctt attataaact gtaagaaaaa 27600

aaaggcccct acttcgtccc ttttgcaaat ctgccttttc ctactcacta accagctggt 27660

tcagagcaag gacactctgt ttggtgccat cgctgcagac tggaaggaag aggtccttgc 27720

cccacaccca acagtctcct gctgttaccg gcaggttggc aggcaggcag gcgagaagca 27780

gccagggctg gtggtgtgtc cagtttgaag actagtttcc agccctggcc ctgctcaccc 27840

tccaagtggc cctggcaggt tcctctacca catcgtggac ttcaccttcc ttctctaaga 27900

agctcaatcc ccaaggcctc attcccatag gccttctcac cctttttctt tccctctggc 27960

tgaatgtggc cagcacgggc ttccaaggcc atcaactcgt ctgcagcagc cccatgcctt 28020

gcagggcctc agagcttcct cctgcctatg acagtgtggt tttggttccc acacttggga 28080

tcagattgaa actcgcctcc gtggtgagaa tatgggacat agagcctcgg tgaccttggt 28140

gagcagcagt ccaggccacc tgctcagcct ggggttgggg ggggctcctc ctccttgact 28200

ggtccttgca tttgcctcca tccagcctgt ctgggctctc cgaggcaatg gagaccagca 28260

ggagtcacga tgggtcagga gccccctttg ggcctcagcc ctgccctgcc ccctaaagta 28320

gcacttggat aagcaaataa attattatac ttactattta tgggtgtggt gaatgggatg 28380

gcaaaggcca agtcttactg atcaccaaac cttaagatat atcctggcag ctagtagacc 28440

cttgggctaa atgaacagaa aactggacaa ataaagtgta cacaaataac tcaaagctgt 28500

catttgtaca cttttcgtct tttcctacta cagtttacat ttttataaag gtgagtagat 28560

ttctaaaatc ccgtggtagg ctctcttgag tttttcttgt atccctgaag ttcagctaca 28620

aataagctaa tcactaacat ttgttgagca tttactctgt tgtcaggccc cgtgccgagt 28680

gctttaggtt cagaatttca tgtcatcccc acagcagccc taggagatga atgcaattct 28740

tatgtccact tgactgataa ggaagttgag gttcaaagag gctaaatgac tctcccaggg 28800

tcccacagct ggaaagtggc cacagggccc cagctggttt tctagggcag caggcagaag 28860

gcgaggagga tctgggccct gtggtgcccc agcctcatct gagggtcctc atctgagaga 28920

acaggatcct cacagcatgg gcaggctgca agtggtccct gaggttatcg tggagtggac 28980

cctgacttga cctgagtctg tttggacccc agacctgctg aacaaccaca tcttgaagtc 29040

agctatgtgt gctgaagcca tcgttgcggg gctgtctgta gagaccctgg agggcacgac 29100

actggaggtg ggctgcagcg gggacatgct cactatcaac gggaaggcga tcatctccaa 29160

taaagacatc ctagccacca acggggtgat ccactacatt gatgagctac tcatcccaga 29220

ctcaggtagg ccaggcctcc gggggccttg gccctgcctg gcccaccatc tcttctgcca 29280

tcctttgtgg cgggggaggg gaaattcaga gatctttggg cgacttccct gcctggaccc 29340

agctcacagc ttctcggcca ctgcaaatgt gtgggttgtg accagactga tgtgtcttga 29400

gcttcaggct tgcaagtgca gtggagaggc agtggggagc tattgaaggg gtctggggac 29460

agactcaatc acagaggcct ttcagaagat ctgcctgctg tgcatgggca aagagggcca 29520

cttgctgacc tcagagcatg tgctttctca gtagtgccca agctgtccca tggtcactga 29580

cccagttaga atgactgaat ggactttggc ttgtgtctca ttaggaatcc tagccccatt 29640

ctagtcttcc agtgagatct gtccatgagt gaaggaatct cacaggaaaa aacaaaatgc 29700

ttctatgggt gtggttgctg gccttatcta caccacagaa gccatcacac agactgtctt 29760

tcttcccatt gttagaatgt gccctgacca agcagcccac agggcctggg acagaggctg 29820

atctctgcct aactgagctc acctctcctc cctctcctcc tgactggtta gattttctag 29880

gtgactgttc ccctgatgac acaagcccgc tgggccccag cagtgtttag aggggttgtt 29940

gactcacgag atgacattcc tgctgatgtg tgtcatgccc tggggtggat gaatgataaa 30000

tgaaaacagc gcttttaact tttgaaccca ctttctcctt ccttgtagcc aagacactat 30060

ttgaattggc tgcagagtct gatgtgtcca cagccattga ccttttcaga caagccggcc 30120

tcggcaatca tctctctgga agtgagcggt tgaccctcct ggctcccctg aattctgtat 30180

tcaaaggtaa catggggaag gcatccctgt tagattgtcc ctggaggcag cttccccacc 30240

cctgtcacct ccacaacact ctccgattta cagcacccca tgggacatta gaacttccac 30300

tcagctcaac caaaagcaga tgtgacttca gcagaaactt cagaggctct gttgtttcat 30360

taggcagtgc agagaatgcc tttggggagc cgttcctcag aactcaagac ttgacatctg 30420

ggaggcagcc gttcctcaga actcaagact tgacatctgg gagagcagag cattcccttg 30480

cctttctatt tgcagggtca cttgccaatg tatagtcaag aggtcagagt gagggtacag 30540

ctgagctgca gccccaggaa ggcagagaag ggggccaagt tgtgtgcgtg cctgcccttc 30600

cctcttaggg caaaactcca aacacccttg attatctgga tcttctttaa ttctccatag 30660

aagataccag atgttaagga atattggcag cttcacttgg tttctcaatc cctgtttcca 30720

aactcaagga gggatgggct ttttcactgt atttatctct catcactctc ttcattgcag 30780

gagcacatct ctctggacct aaccatcacc ctttcttgta gatggaaccc ctccaattga 30840

tgcccataca aggaatttgc ttcggaacca cataattaaa gaccagctgg cctctaagta 30900

tctgtaccat ggacagaccc tggaaactct gggcggcaaa aaactgagag tttttgttta 30960

tcgtaatgta agttctgggt cctaaatcat gctcctggga agctccttac tgtgggactt 31020

gtattagtgt aaaaaaaaat gtcctcaata agcaggagtt tgcatgagaa ctggttgctg 31080

acaaggaagg aaataatttc tggaaaatat agataacaaa atgagatcct gcagaaggat 31140

tggaatctct ttttctggag gcctttgaga ataaaccaca caattatcca acctgtattg 31200

tgaaggaata agtccttctt gaattcagga attaacacct gggaggaggg atggagttca 31260

gactctttct gagcttatga gaagagaagc cccctaaact aaaatacagc cctccttggt 31320

ccaaaaggtg ccttctctct tctgctgtat cttctttgtt ttcaaaccca acagttaccc 31380

tggaaatcaa aaaggaagta caactcaaca tagctcttgc ctgggaccaa ccagcaccat 31440

ttggctaaag atggttatca tctgttaaac aaagaaataa ataaatgggt tcaacgtatt 31500

tatttcaaca ttgtcaatgg acctcatgtg taactgatat tctcattatg ggacctctgt 31560

gtgactttat tggggcctct ctaaccgttc tttccttaag gaagaccatt tattgtttta 31620

tttcctggag aaaatacatc attttatccc agccttaata acccatccca gtgtatactc 31680

cttcatcttc atggataatg accctgctac atgctctgaa caaatcagga ggcccctcgt 31740

ggaagtataa ccagtccttt ctttctctgt ccctcttctg tgcagagcct ctgcattgag 31800

aacagctgca tcgcggccca cgacaagagg gggaggtacg ggaccctgtt cacgatggac 31860

cgggtgctga cccccccaat ggggactgtc atggatgtcc tgaagggaga caatcgcttt 31920

aggtaattag ttccatcccc gggtggagct tctgcccagt ggtcatgctg gagtgggatg 31980

tggggcccca gctatttgtc aagctttctt ctaccttggg gattcaatta acactagcag 32040

tgcactgctg cgaccttcca gacttgggat ggggaaaagg caagggtcgc cttgaaagct 32100

tacattggga agaagggtta cttctaagag tgtaatcttc acatgcatgg gaagcaggga 32160

ggggggacta catttttatg actgaagtgc aaggaaaaca tcaccctctc attgtaaagc 32220

tccaagtgag ccaagagcac atagtttaca gtgcacgatg agcctctcac tctctgcgca 32280

gtatctgttt attgcaactg aagcaccctt gtgagtttgt tttcttgccc ggctatctcc 32340

atttctgact tgctcattca ccttggggtg ctgtcatatt gaatgtttcc ctgtcactga 32400

cttcagccac ctgcacaagg gcttggagac cacacccctc tgccctccca gaatcatatc 32460

cctggaggct cagctagtct ctgggtcagc catacctctg ccctttcttt tccctccttt 32520

ctcctgtggc ctctgacgtc tggccattta acagagctta gcatttttgc tgggtggaga 32580

gagctggagc ctggaatcac tccctctttg tgcatacgga gggcatgaaa accaaggtgt 32640

gtgcattcca gtggcctgga ctctactatc ctcagtggtg aggtatttaa ggaaaatacc 32700

tctcagcgtg gtgaggtatt taaggaaaat acctgttgac aggtgacatt ttctgtgtgt 32760

gtatctacag catgctggta gctgccatcc agtctgcagg actgacggag accctcaacc 32820

gggaaggagt ctacacagtc tttgctccca caaatgaagc cttccgagcc ctgccaccaa 32880

gagaacggag cagactcttg ggtaaagacc aacttaagta cacgtctcca tttttctaaa 32940

gtagtgatcc ctcagggccc cagcagcaaa cagttggcac atcaaggatt gacttgaagg 33000

gattttatga caagactatt agtgaaagag tgggcgggac taaaggaact agcaaaggat 33060

gaggccaacc agggactagc aaccctggga agcctttact acccctaggc ctgggggaat 33120

gggaggatga gagcaggaac cagggaggtc atgagccttg gacaagggca cagaacagca 33180

gccagagcca tgtgcagcca gccactgtca gaaccatgca agggggacca ctcagcgccc 33240

cagcctccct ctcagacagt tgccatctgg gtctcttgtt ggctgatgcg agagcaggag 33300

ggagcccact gatgcagttc atagagctca gcctcctggg caggaaaccg ggcagagagg 33360

agtagaaaag aattaagggt ggctgcgacc agcccagtca ctgaggcacg tttcccactg 33420

gagacctatg agcacagtga taataaagcc agttacctgc actgactatc cctccagaca 33480

aaagctttcc caagaagtta gtcatggctc tgagagatct agttgaggat gtttggcagg 33540

ggatctagtg gttacgggtg gctaagaaaa atgaggaagg taagagtatc ttgcagcctg 33600

tgttgggagg attaaatagg atgccacaca cagggccagg cagacagcct ggtcagtaat 33660

agccatgacg atgggggcgg ggggagcagg aatgggagttgcagtgttta gctcagatgc 33720

atgcctgtga gagatgcttc cactctcaca gaaagatgag accaaggaaa aggaggagga 33780

agaggaagga ccttgacaaa ccttggggcc cacattgtct acacctccct tcctgctcta 33840

gagcagaata gaaagttcag gttgcaggca gctctaagtt gaattcgtgt cctgtttaat 33900

tttctttatt gctaaatgaa tgcctgtgtc tgtgatgctg acgtatgttc ctaaggagag 33960

gggagaagtt cattctgaac ataaactttt catcctctct ctgtccagca agaatggaat 34020

attccccaag tggcctgagc cagcttggct ttctttttgt tttcaattat gtgggagttg 34080

aggaggggga tgggaaaagc ttcccaaaca caccctcccc caggcctgag gcacccctgg 34140

gggacagaga gtgttagagg ttggtacagg tgttagagat attgaaagga catcccatgc 34200

accccagggg ctggtgtggc tctgtacttc caggcaatat tttgtggaag gggaaccttg 34260

tcagctccag gttgtggatg tttgaaaatc agttggtacc cagtggctcc atcctctggc 34320

aggcatgtgg atttgtcaat aaccaagtga actctccaaa ataagttaaa acttcctccc 34380

ttctcagttt caagatgctg gaaatagctg ttcataagcc ctggggaaat ttagcccttt 34440

ggctggtaat gggagtatcc gagatgagag ggcagctgga aactttcgga atgacctccc 34500

acacttaatt tgggaaatgc ctctgcacct ttatgggcaa ccagatgcct gccccagttg 34560

ctggagacac tgatgtgggc tgaaaggaat gctgagacgt gacgaggaga gatgctgcgg 34620

agggaatatc cccctcagcc ctgacctcat cggctccatg gctcctccac agtacagctg 34680

tctactcttt taagttctcc cttcaggaaa tagccatctc aaacagaatg tgcatttgag 34740

ggcagaatgt gtaaatattg cactactgtg ttataaccgt caggagccat gctgatgatg 34800

aaacgtccca gatgccggtg ctggaaaggt ccctggcttt ccaagcaaat atttatctca 34860

tggaaacatg agtcatactc acagaggagt atggattaac tccttctcag cagccaggga 34920

gcccagcatc ccagacagca tatttaaccc agaggccaac tgactgctgg ggcagatttg 34980

tggtcatgaa catgtgcttt gtgtcctctg accattagac agattgtggg tcacaacgtt 35040

gagtatacag tgggagctta ataagtgctt attccctggg cagggagttc ttcatttcag 35100

gggtgaccac ttacatcttc tcctctgggc cctccttgac caggctaatt accattcttg 35160

ggattaactc tatctccttt tcccgcaacc tgcaggagat gccaaggaac ttgccaacat 35220

cctgaaatac cacattggtg atgaaatcct ggttagcgga ggcatcgggg ccctggtgcg 35280

gctaaagtct ctccaaggtg acaagctgga agtcagcttg gtaagtgtcc tgcaaatcaa 35340

aggctggcta aatttcccca gggcagggct ccaggacata tctcaccccc aggatggaat 35400

tatacacaca caaccttcaa gttgcagccc gaatctctga gtgtaattcg tccaaagaaa 35460

aagagaaaag agaagagggt cttcagggaa atcaagtgag atcatagtta gacatgagta 35520

agaacttcca gatttacaag ggaatagagc atctgatttg gcatctgaga gaggctatta 35580

gatcttcctt ctcttaagga ggttgtaggc aactagttat gtgactgaag agatcagtct 35640

gtactcacac catcccaccc cccaaaccca gggcttcact gagttgtacc atgaaccaga 35700

ccatcccaag aggctttttg agttctgaca cttgctctgt gagccttccc ttgctctgca 35760

cattgatgat ataactttgt aactgcacta agagtgttcc taaagcagat agccagccga 35820

gctccagaaa tctccctggc tgcacctgca gaggccactg acccctctgt ggagggaccg 35880

ctcttcagtg tgtggctggc ttctactctc tgctcctctc tcttggtctt cagccatcca 35940

ttgctcacca gtttctcacg aggagcatag gaagatatgc atgtagggag gtaggcacgg 36000

ggatgacttg tttgacttta gcaggtcatt caagaatctc ctcgcacctg gtttcagatg 36060

ctggggtcct gtctgtcaca ggcttctgtg cctcctaccc ccttgagttt gtcacatggc 36120

ccttcaggaa ggcctgagat agatttgccc tgggtgggcc tcctatgaga aaatcttaag 36180

tgaggcaccc aggcaaaatg gaaagagcct tttgcccaga gcaggaagcc tgtcttccat 36240

ttccagctgt tccacctact tagcttaaaa gaggcacttc gcctgtcttc agtctcagtc 36300

tcagtctcct cttctgtgga atgggacaat aatatctact ctccttatca tacactgctg 36360

tgaggactga gtggatcaca caaaaaagca ttatgtaaat tgcaaagtgc taaatccaca 36420

caggagattt gaattaatcc accacactga aggtctgtca agggcaggga ctgtttcatt 36480

caccagagta tccccagtct aacacaggac ttggcatatg aaaagtgttc agtaggccgg 36540

gtgcagtggc tcatgcctgt aatcccagca ctttgggagg ccaaagtggg cggatcatct 36600

gaggtcagga gttcaagtcc agcctggcca acgtggtgaa accacatctc tactaaaaat 36660

acaaaattag ctgggcgtgg tggcacatgc ctgtaatcac agctactctg gaggctgagg 36720

caggagaatc acttgaaccc aggaggcgga ggttgcagtg agtcgagatc atgccactgc 36780

actccagcct gggcgacaag attgaaactc catctcaaaa acaaagaaca aggaaaaaaa 36840

cgaaaactgt tcagtaaaca cttgctgaat gaataaaata aatatataaa tgtataaata 36900

aatgctctac tttcaaccac tactctgtttttcttttaga aaaacaatgt ggtgagtgtc 36960

aacaaggagc ctgttgccga gcctgacatc atggccacaa atggcgtggt ccatgtcatc 37020

accaatgttc tgcagcctcc aggtaagtgt cgcatcccca ctgactctgc agccagtcct 37080

tttcttcatg tggcagttgg tggagagaag aaaaactgtt ctaaacaatg atgagaataa 37140

catgtaattg tgatagttaa actgtgccta tgtgactgat tgcagagtga attgggagct 37200

gttggttttg aatgcaccac actaaggaat gtgaggacac attgctcttt gcggagttgc 37260

ccagctatat tagctcccct cggacacagc ccagttttct gtattcgcgt ggatgctgtc 37320

cgcgcgattc ccagcactcc tcttacagca tctcacctca gtgtatgttc cttgcctcca 37380

gtgcagttga acctcagtcc tgcctctcct catgtgtgca ttcacctttc ttggtgctct 37440

ctccccatgg gccaagttct accatgagtt atgaaacatt atggagaaaa catgtctttg 37500

gaaatgtgag ccagaaagcc caccagtgcc cctcagtcac ggttgttatg aatgacatgc 37560

taatggtttc actctggtca aacctgcctt ttctttcctc ttcagccaac agacctcagg 37620

aaagagggga tgaacttgca gactctgcgc ttgagatctt caaacaagca tcagcgtttt 37680

ccagggtaag atgcctgcta ggtttgcgcc tagcctgagc agcctcaggt cctctgtttg 37740

ggccatagag gagcctctcc agcccctgtc ttccttggct gctccccagg gctctcttaa 37800

aacttctccc cactcccact gaggcatcct cagccccagc ctgtgtcaaa ttcagagtaa 37860

agaaccaagg caactccctg gctttcatgg gccaaagcgc aggctttcac accgaggcct 37920

ctgagcctca gatcatgggg aagtcactgc tggagagaac agacatagct ctggaagcca 37980

tctgcccaag agggcagccc atcccaagtt catcttacag tggccaggcc tgccctgagc 38040

cggggcctct gggtcactct tctgctgtcc atggcattgc ccatcctggg tgaggctggg 38100

gctctcctgg gcactgtatg tattctggat acagggatac tgggctcgct atgtgtgtgg 38160

agccatccct tccttgcccc agccccacct ccctctcaaa ccctctctgg ctctttctga 38220

gcttcctttc ctgctcccca gcttgcccag tgctcagtgc cccacttggc tcttttgcta 38280

cttcgggtca ggtggagcct cttgggaatg tgaagtgcct tacagaaaga ttgcacttca 38340

agaggagagg ctgcagggag ccatcctaaa cccagaggcc tggagcttac tgtgtcactt 38400

tacttttgta cacaggggtc tccttagtgc cctcgagaag gattcttggc cctgagcttc 38460

tactcctgag gccacctctg tgcagcccca gctccctcaa ctctaggctg tagtctcagt 38520

gggaaagcct ggcttggggg tctcctagga atgtccacct gaaggcacac ttgatagggg 38580

cttgcacaac ttatgtctgc caaggccacc tgaggaactc cctggtgcct ataagttcca 38640

ccttcccctt cctcttcctc gccccagcat tttttctgag taggggtggc aatgggcaaa 38700

gccattgtca taagcagttg caggtataac tttcactaga aaacctgaca ccttgtgttt 38760

tctttcaggc ttcccagagg tctgtgcgac taggtgagtc tggtctgggt ttgaagtcat 38820

tgcagacctg tttaggcctt acccccaagc aagcccaagc ctgccatctg ctgtatatag 38880

ataagaacat catggtgcag taaaagaagc ctggcctttg gagtcagaac agcagggtga 38940

cttggggtca gacccagagc accccatttc cttctctgta agatgaggat aataagagta 39000

acaacctttt agggttaagg tgagttttca gcttaggaag tctgggaata ttgcaaaggg 39060

cttggcagga acccatggtg aggatctagt tccaagttga taggtacaga aaaccagaac 39120

atcgggcctt gagtaaagag tgaagtttca caaaccacaa agcacctgct atgtgcagga 39180

gagcatggca gaaggaggct gcttggccct ggtccttgag attctgacag tgtcctagac 39240

agacatgggg agatctgcac ctatttgacg ttaccaactt ctctttttca gcccctgtct 39300

atcaaaagtt attagagagg atgaagcatt agcttgaagc actacaggag gaatgcacca 39360

cggcagctct ccgccaattt ctctcagatt tccacagaga ctgtttgaat gttttcaaaa 39420

ccaagtatca cactttaatg tacatgggcc gcaccataat gagatgtgag ccttgtgcat 39480

gtgggggagg agggagagag atgtactttt taaatcatgt tccccctaaa catggctgtt 39540

aacccactgc atgcagaaac ttggatgtca ctgcctgaca ttcacttcca gagaggacct 39600

atcccaaatg tggaattgac tgcctatgcc aagtccctgg aaaaggagct tcagtattgt 39660

ggggctcata aaacatgaat caagcaatcc agcctcatgg gaagtcctgg cacagttttt 39720

gtaaagccct tgcacagctg gagaaatggc atcattataa gctatgagtt gaaatgttct 39780

gtcaaatgtg tctcacatct acacgtggct tggaggcttt tatggggccc tgtccaggta 39840

gaaaagaaat ggtatgtaga gcttagattt ccctattgtg acagagccat ggtgtgtttg 39900

taataataaa accaaagaaa catacgtcct gtgtgcatgg tacagtgtgc tgacctgagg 39960

ccgtcatgct cctccacacc tcaattctgc tctggagaag ctcagaaagg agccccgagg 40020

gatggttttg gggagattcc agcagccagc cctcagacag ccagacagct catgggggtt 40080

tgagcctgtc tttgccaaac aggtttttat ttcaccctcc tccggtcctg gggtttcaag 40140

ttttcagtgt tgccttcacc ccgcacttta ttcctcttat tacttggaag taccttccct 40200

ccagcatggt gatcccctgc ctgtgtgctg gacttttgag tcctcagcac caacctgtga 40260

agtggttgcc agcataatcc cattatgcag atgaggagac caaggcccag ggaagggaga 40320

accaccagca gcacgtaaaa tagctgagct gggactggaa ctcacacctc ctgactctca 40380

gtgaccacca ctgacaacag cataagtcca ggttttccag gcccatcccc tctgtgccaa 40440

cccacattca gattccttcc ccggctcccg taatctctgg catctagaat atcctcagga 40500

ctctgagagg tgatatcatg tggttgtggt gccattgccc cctacctgtg tggcctgggg 40560

ccagtcatgt gacctcccag ggtctcctct tctgtaatag ggagatgacc gtcacatcta 40620

cttcatgggt ccatcgtgag gatgaaatga gatgatctat ataaaatgct tggtacaaca 40680

ttaggtggcc ttatttttat cctgccgtct gggactgctc aggatcaatg cgccagagag 40740

cctttatttg tgtctttccc acaggtgggc tggcccactt tcctagagaa tgggacagac 40800

ctccttccca cccacaccca tctctgccaa ggctgattca ctccagcagg cggagctcat 40860

ttcacttcat ggaaccaatg acccaaagat atatccccag cactactgct ggtcagtcca 40920

ctgctgctgg gaatacagca atggtagtgg cagacagagg ccctctctta aatagcttcc 40980

agtctgagga aagagagata tgacatcaat ccattaaaat cattcatcca ttggttccac 41040

aaatatttgt tgagggctac ctatgtgcac ccccatgtta gaccctgggg aatagacatg 41100

tcattctcat gaggcttctc tactgatggg ggggaagaga attgtcaacc agataatggc 41160

actacagcct gtgtgttctt agtgactctg aggatagcac tgtggttctg tgacagataa 41220

tgaaggattt ggaagcagga atgcccagga gctcccagaa gtgggaagag atgagaggaa 41280

tggaaggaac ttacctgaag gtgaaggcat caggctaggg gaccaaggga gaaggtgtcc 41340

tgagaggtaa ggcttaacct tgggtgtgaa ttcagttccc gtcactctcc catagctctg 41400

tcctgctgtt cccacctccc ctgcagccat gcgggcttgg gcggctagtg agggccttgc 41460

tcatgctggg tatcctatgc tatgcttcac tttgagcacc taaaatacac acactgcact 41520

ttaccaagat gacctcggaa accaaagagg tgatcagcat aagttttaaa gacccttaaa 41580

tttaaagtaa aaatcactac aggatccatt ataaatgcca aacactaaga tgtgtgtttc 41640

cagttctccc cttcatttgt ccctgccact ccctgccctg actttgcccc accccctagt 41700

aatgtgggct ccactctatg ctccaaactc tccctggaga gaaatcctcc ctgtggttga 41760

ggacaaggcg cagccttccc ctcccaccaa agaaggtcag attccctttt ttggttccta 41820

accatccata ccccttcttt tctcatgaag actcgggcta agcattcatt agggctgcca 41880

tctggaggat ggacccttag agctgagggg ccagcactgt gtgt 41924

<210>62

<211>683

<212>PRT

<213> Intelligent people

<400>62

Met Ala Leu Phe Val Arg Leu Leu Ala Leu Ala Leu Ala Leu Ala Leu

1 5 10 15

Gly Pro Ala Ala Thr Leu Ala Gly Pro Ala Lys Ser Pro Tyr Gln Leu

20 25 30

Val Leu GlnHis Ser Arg Leu Arg Gly Arg Gln His Gly Pro Asn Val

35 40 45

Cys Ala Val Gln Lys Val Ile Gly Thr Asn Arg Lys Tyr Phe Thr Asn

50 55 60

Cys Lys Gln Trp Tyr Gln Arg Lys Ile Cys Gly Lys Ser Thr Val Ile

65 70 75 80

Ser Tyr Glu Cys Cys Pro Gly Tyr Glu Lys Val Pro Gly Glu Lys Gly

85 90 95

Cys Pro Ala Ala Leu Pro Leu Ser Asn Leu Tyr Glu Thr Leu Gly Val

100 105 110

Val Gly Ser Thr Thr Thr Gln Leu Tyr Thr Asp Arg Thr Glu Lys Leu

115 120 125

Arg Pro Glu Met Glu Gly Pro Gly Ser Phe Thr Ile Phe Ala Pro Ser

130 135 140

Asn Glu Ala Trp Ala Ser Leu Pro Ala Glu Val Leu Asp Ser Leu Val

145 150 155 160

Ser Asn Val Asn Ile Glu Leu Leu Asn Ala Leu Arg Tyr His Met Val

165 170 175

Gly Arg Arg Val Leu Thr Asp Glu Leu Lys His Gly Met Thr Leu Thr

180 185 190

Ser Met Tyr Gln Asn SerAsn Ile Gln Ile His His Tyr Pro Asn Gly

195 200 205

Ile Val Thr Val Asn Cys Ala Arg Leu Leu Lys Ala Asp His His Ala

210 215 220

Thr Asn Gly Val Val His Leu Ile Asp Lys Val Ile Ser Thr Ile Thr

225 230 235 240

Asn Asn Ile Gln Gln Ile Ile Glu Ile Glu Asp Thr Phe Glu Thr Leu

245 250 255

Arg Ala Ala Val Ala Ala Ser Gly Leu Asn Thr Met Leu Glu Gly Asn

260 265 270

Gly Gln Tyr Thr Leu Leu Ala Pro Thr Asn Glu Ala Phe Glu Lys Ile

275 280 285

Pro Ser Glu Thr Leu Asn Arg Ile Leu Gly Asp Pro Glu Ala Leu Arg

290 295 300

Asp Leu Leu Asn Asn His Ile Leu Lys Ser Ala Met Cys Ala Glu Ala

305 310 315 320

Ile Val Ala Gly Leu Ser Val Glu Thr Leu Glu Gly Thr Thr Leu Glu

325 330 335

Val Gly Cys Ser Gly Asp Met Leu Thr Ile Asn Gly Lys Ala Ile Ile

340 345 350

Ser Asn Lys Asp Ile Leu Ala ThrAsn Gly Val Ile His Tyr Ile Asp

355 360 365

Glu Leu Leu Ile Pro Asp Ser Ala Lys Thr Leu Phe Glu Leu Ala Ala

370 375 380

Glu Ser Asp Val Ser Thr Ala Ile Asp Leu Phe Arg Gln Ala Gly Leu

385 390 395 400

Gly Asn His Leu Ser Gly Ser Glu Arg Leu Thr Leu Leu Ala Pro Leu

405 410 415

Asn Ser Val Phe Lys Asp Gly Thr Pro Pro Ile Asp Ala His Thr Arg

420 425 430

Asn Leu Leu Arg Asn His Ile Ile Lys Asp Gln Leu Ala Ser Lys Tyr

435 440 445

Leu Tyr His Gly Gln Thr Leu Glu Thr Leu Gly Gly Lys Lys Leu Arg

450 455 460

Val Phe Val Tyr Arg Asn Ser Leu Cys Ile Glu Asn Ser Cys Ile Ala

465 470 475 480

Ala His Asp Lys Arg Gly Arg Tyr Gly Thr Leu Phe Thr Met Asp Arg

485 490 495

Val Leu Thr Pro Pro Met Gly Thr Val Met Asp Val Leu Lys Gly Asp

500 505 510

Asn Arg Phe Ser Met Leu Val Ala Ala IleGln Ser Ala Gly Leu Thr

515 520 525

Glu Thr Leu Asn Arg Glu Gly Val Tyr Thr Val Phe Ala Pro Thr Asn

530 535 540

Glu Ala Phe Arg Ala Leu Pro Pro Arg Glu Arg Ser Arg Leu Leu Gly

545 550 555 560

Asp Ala Lys Glu Leu Ala Asn Ile Leu Lys Tyr His Ile Gly Asp Glu

565 570 575

Ile Leu Val Ser Gly Gly Ile Gly Ala Leu Val Arg Leu Lys Ser Leu

580 585 590

Gln Gly Asp Lys Leu Glu Val Ser Leu Lys Asn Asn Val Val Ser Val

595 600 605

Asn Lys Glu Pro Val Ala Glu Pro Asp Ile Met Ala Thr Asn Gly Val

610 615 620

Val His Val Ile Thr Asn Val Leu Gln Pro Pro Ala Asn Arg Pro Gln

625 630 635 640

Glu Arg Gly Asp Glu Leu Ala Asp Ser Ala Leu Glu Ile Phe Lys Gln

645 650 655

Ala Ser Ala Phe Ser Arg Ala Ser Gln Arg Ser Val Arg Leu Ala Pro

660 665 670

Val Tyr Gln Lys Leu Leu Glu Arg Met Lys His

675 680

Claims

1. A reaction mixture for detecting corneal dystrophy in a subject, the reaction mixture comprising a labeled probe comprising a sequence selected from the group consisting of SEQ ID NOs: 25-30, 36 and 54.

2. The reaction mixture of claim 1, further comprising a corresponding labeled probe comprising a sequence selected from the group consisting of SEQ ID NOs: 19-24, 33 and 50.

3. The reaction mixture of claim 2, wherein the labeled probe is selected from the group consisting of SEQ ID NOs: 25-30, 36 and 54; and/or the corresponding labeled probe consists of a sequence selected from the group consisting of SEQ ID NO: 19-24, 33 and 50.

4. The reaction mixture of any one of claims 1 to 3, wherein the reaction mixture comprises a labeled TGFBI G623D probe comprising the nucleotide sequence of SEQ ID NO: 36; and a labeled TGFBI M502V probe comprising the sequence of SEQ ID NO: 30.

5. The reaction mixture of any one of claims 1 to 4, wherein the labeled probe is fluorescently labeled.

6. The reaction mixture of any one of claims 1 to 5, wherein each of the labeled probes comprises a different probe and is independently labeled with VIC, FAM, ABY, or JUN.

7. The reaction mixture of any one of claims 1 to 6, further comprising at least one amplification primer pair for amplifying TGFBI gene sequences from a biological sample from the subject.

8. The reaction mixture of any one of claims 1 to 7, further comprising:

(a) a corresponding forward primer comprising a sequence selected from SEQ ID NOs: 7-12 and 41; and

(b) a corresponding reverse primer comprising a sequence selected from SEQ ID NOs: 13-18 and 47.

9. A reaction kit comprising (a) a reaction mixture according to any one of claims 4 to 8, and (b) one or more labeled probes for one or more TGFBI mutations selected from R124S, a546D, H572R and H626R, separate from the reaction mixture.

10. The reaction kit of claim 9, wherein the one or more labeled probes are selected from the group consisting of seq id NOs: 19. 25, 20, 26, 21, 27, 23, 29, 50 and 54.

11. The reaction kit of claim 9, wherein the reaction kit comprises a labeled TGFBIR124S probe comprising the sequence of SEQ ID NO: 19 or 25.

12. The reaction kit of claim 9, wherein the reaction kit comprises a labeled TGFBI a546D probe comprising the sequence of SEQ ID NO: 20 or 26.

13. The reaction kit of claim 9, wherein the reaction kit comprises a labeled TGFBI H572R probe comprising the sequence of SEQ ID NO: 21 or 27.

14. The reaction kit of claim 9, wherein the reaction kit comprises a labeled TGFBIH626R probe comprising the sequence of SEQ ID NO: 23. 29, 50 or 54.

15. The reaction kit of claim 8, further comprising a third amplification primer set.

16. A method for detecting corneal dystrophy, comprising:

(a-1) amplifying a first TGFBI gene sequence from a biological sample from a subject using a reaction mixture comprising at least a first amplification primer pair and a set of at least two detection probes;

(B-1) hybridizing the first detection probe and the second detection probe in the set of at least two detection probes with the first TGFBI gene sequence having the G623D mutation and the second TGFBI gene sequence having the M502V mutation, respectively; and

(C-1) detecting one, two or more mutations in TGFBI gene sequence based on hybridization of the first detection probe and the second detection probe to the first TGFBI gene sequence and the second TGFBI gene sequence, respectively.

17. The method of claim 16, wherein the reaction mixture comprises the reaction mixture of any one of claims 1 to 8.

18. The method of claim 16 or 17, further comprising:

(a-2) amplifying a third TGFBI gene sequence from the biological sample, wherein the reaction mixture further comprises a third label probe for a third TGFBI mutation selected from the group consisting of R124S, a546D, H572R and H626R;

(B-2) hybridizing the third label probe with the third TGFBI gene sequence; and

(C-2) detecting a mutation in the third TGFBI gene sequence based on hybridization of the third detection probe to the third TGFBI gene sequence.

19. The method of any one of claims 18, wherein amplifying the first TGFBI gene sequence (a-1) and amplifying the second TGFBI gene sequence (a-2) are performed separately.

20. The method of claim 18, wherein said hybridizing (B-1) and said hybridizing (B-2) are performed separately.

21. The method according to any one of claims 18 to 20, wherein the detection (C-1) and the detection (C-2) are carried out separately.